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Our initial models (see Computational Methods) investigated both anti and syn conformations of the adduct in the active site of Dpo4, partnered with dATP or unpaired in the −1 deletion simulations. In addition, anti -G* opposite anti -dCTP, anti -dTTP or syn -dGTP were investigated using results from dATP to guide selection of the starting structures. Production MD simulations were carried out for 3 ns and the ensembles of structures derived from the last 2.5 ns trajectory were analyzed.
|
16820532_p16
|
16820532
|
Initial models
| 4.14409 |
biomedical
|
Study
|
[
0.9993783235549927,
0.0002812990569509566,
0.00034033210249617696
] |
[
0.9995635151863098,
0.00017686410865280777,
0.00020389985002111644,
0.00005572180452872999
] |
en
| 0.999995 |
A number of structural criteria were evaluated in each modified system and compared to the undamaged control simulation. These include: (i) the number and occupancy of the hydrogen bonds in the nascent base pair ; (ii) the distance between C1′ of the template and C1′ of the incoming nucleotide (dNTP), normally near 10.8 Å in a Watson–Crick pair ( 65 ) (Supplementary Table S7); (iii) stacking interactions between the nascent base pair and the primer-terminus base pair ; (iv) the frequency of sampling a near reaction-ready distance (3.1 to 3.5 Å) between O3′ of the primer-terminus and Pα of the dNTP (Supplementary Table S7); (v) angle O3′ (primer 3′ end)-Pα(dNTP)-O3α(dNTP), ideally 180° for in-line attack of O3′ on Pα ( 66 ) (Supplementary Table S7); (vi) the chelation of Mg 2+ ions ; (vii) the distance between the two Mg 2+ ions (Supplementary Table S7) and (viii) hydrogen bonds between the nascent base pair and the polymerase. These are summarized in Tables 1 and 2 , using a distortion scoring function to evaluate the lesion impact on the polymerase active site ( 34 ). While we analyze the structural features individually, we realize there is interdependence among them; how they affect each other remains unclear but their composite quality sheds light on a given model's structural feasibility. While scoring has subjective criteria elements, it reflects our current stage for evaluating polymerase active site distortions. We hypothesize that the more distorted the active site is, the less efficient the nucleotidyl transfer reaction will be.
|
16820532_p17
|
16820532
|
Structural analyses
| 4.301758 |
biomedical
|
Study
|
[
0.9993523955345154,
0.0003930744423996657,
0.0002546081959735602
] |
[
0.9991472959518433,
0.0002518848341424018,
0.0005150791839696467,
0.00008583086309954524
] |
en
| 0.999997 |
Unmodified control of anti-G·anti-dCTP. In the unmodified control simulation, anti -G Watson–Crick pairs with an incoming anti -dCTP. Views of this complex and its active site after 3 ns production MD simulation are shown in Supplementary Figures S3a and S4a, and Figure 2a . The active site remains essentially undisturbed throughout the simulation. The Watson–Crick hydrogen bonds in the nascent base pair have occupancies >90% (Supplementary Table S5); the C1′–C1′ distance in the nascent base pair remains normal (10.9 ± 0.1 Å); the chelation of the two Mg 2+ ions is preserved ; the distance between the two Mg 2+ has an average value of 3.7 ± 0.1 Å; the Pα–O3′ distance samples the near reaction-ready range frequently, during 78.7% of the time; and the in-line attack angle is 167.1 ± 5.1°, close to the ideal value of 180°. These active site features are similar in polymerase ternary complex crystal structures ( 67 , 68 ). In the active site of Dpo4 polymerase, the flat face of the nascent base pair is topped by protein residues with unusually small and hydrophobic side chains: Val32, Ala42, Ala44, Ala57 and Gly58 . These residues form the ‘ ceiling ’ of the dNTP binding pocket ( 25 ). The ‘ floor ’ of this pocket is considered to be the flat face of the base pair at the primer terminus ( 25 ). The geometry of the dNTP binding pocket remains normal here . A highly conserved aromatic residue in the DinB family (Tyr12 in Dpo4, Phe12 in Dbh and Phe13 in DinB) is tightly packed on the sugar ring of the dNTP. This has the effect of placing the dNTP properly for catalysis and acting as a ‘steric gate’ to prevent binding of ribo-NTPs through steric exclusion of a 2′-OH ( 32 , 69 ). In our unmodified control simulation, the proper position of Tyr12 is preserved. Polymerase residues Tyr10, Phe11, Tyr12, Ser34, Thr45, Arg51, Lys159, Thr250 and Arg331 form hydrogen bonds with the nascent base pair in the unmodified control simulation (Supplementary Table S6).
|
16820532_p18
|
16820532
|
G·dNTP models
| 4.517421 |
biomedical
|
Study
|
[
0.9987829327583313,
0.0008907434530556202,
0.00032634095987305045
] |
[
0.9987523555755615,
0.0004979399964213371,
0.0005237538716755807,
0.0002259881148347631
] |
en
| 0.999995 |
Syn-G*·anti-dATP minor groove. The syn -G*· anti -dATP minor groove model is highly disturbed. With G* syn , the PhIP rings are on the minor groove side and inserted between the nascent base pair and the base pair at the primer terminus . The location of the PhIP rings in the syn -G*· anti -dATP simulation disrupts the flat face of the primer-terminus base pair, and also causes poor stacking interactions between the nascent and the primer-terminus base pairs, as shown by energetic consideration: a partial energetic assessment of differential stacking interactions can be obtained from van der Waals interactions between bases for the same sequences. In the syn -G*· anti -dATP simulation, this interaction between the nascent and the primer-end base pairs is only −2.3 ± 0.5 kcal/mol, significantly weaker than −16.8 ± 0.9 kcal/mol in the anti -G*· syn -dATP, or −15.8 ± 0.9 kcal/mol in the anti -G*· anti -dATP case. The syn -G*· anti -dATP also manifests an enlarged C1′–C1′ distance (12.2 ± 0.4 Å), and a less frequently sampled near reaction-ready Pα-O3′ distance (59.8%). The hydrogen bond in the nascent base pair in the syn -G*· anti -dATP initial model, between O6 of the syn -G* and N6 of the anti -dATP, is ruptured during equilibration and remains broken throughout the simulation. To achieve the hydrogen bond in the initial model, the PhIP moiety was positioned in a crowded region near the ceiling of the dNTP binding pocket, which proved unfavorable . These disturbances, triggered by the syn -G* adduct, are essentially independent of the specific dNTP.
|
16820532_p19
|
16820532
|
G·dNTP models
| 4.610344 |
biomedical
|
Study
|
[
0.9986265897750854,
0.0010371726239100099,
0.00033631737460382283
] |
[
0.9978558421134949,
0.0009732956532388926,
0.0008329064585268497,
0.0003379815898369998
] |
en
| 0.999996 |
Anti-G*·anti-dATP and anti-G*·syn-dATP major groove. The anti -G*· anti -dATP and anti -G*· syn -dATP major groove models are more disturbed than the unmodified control, but less than the syn -G*· anti -dATP minor groove model. In the anti -G*· syn -dATP complex, the phenyl ring of the PhIP is directed towards the 3′ end of the template, along the template backbone , while in the anti -G*· anti -dATP complex, it is oriented toward the 5′ end of the template . A ∼180° difference in α′ is responsible for this difference in orientation . With an incoming syn -dATP, one full and one bifurcated hydrogen bond are formed in the nascent base pair , with a close to normal C1′–C1′ distance (11.1 ± 0.2 Å). On the other hand, with the incoming nucleotide anti , the C1′–C1′ distance is significantly enlarged (12.7 ± 0.2 Å), and the full hydrogen bond between O6 of G* and N6 of anti -dATP has an occupancy 20% lower than for syn -dATP (Supplementary Table S5). A bifurcated hydrogen bond is formed between the little finger residue Arg332 and the PhIP rings only in the anti -G*· syn -dATP simulation (Supplementary Table S6). The frequencies of sampling the near reaction-ready Pα-O3′ distance and the geometries of the binding pocket are not dramatically different in these two major groove complexes .
|
16820532_p20
|
16820532
|
G·dNTP models
| 4.492986 |
biomedical
|
Study
|
[
0.9989877343177795,
0.0006867123884148896,
0.000325610744766891
] |
[
0.9987439513206482,
0.0004696209798566997,
0.0006065656198188663,
0.00017987711180467159
] |
en
| 0.999998 |
Anti-G*·anti-dTTP and anti-G*·syn-dGTP major groove. The anti -G*· anti -dTTP major groove model is little distorted, while the anti -G*· syn -dGTP major groove structure is very distorted. At this stage, the syn -G* was eliminated due to poor accommodation of the minor groove positioned PhIP regardless of dNTP, as revealed in the syn -G*· anti -dATP simulation . Therefore, only anti -G* was employed to build the models for G*·dTTP and G*·dGTP. With the incoming dTTP anti , the anti -G* forms a wobble pair with the anti -dTTP . During the anti -G*· anti -dTTP simulation, the two hydrogen bonds in the wobble nascent base pair are highly occupied (>94% of the time); the C1′–C1′ distance has a normal average value of 10.8 ± 0.2 Å; the Pα-O3′ distance remains in the near reaction-ready range almost all the time (93.0%), and the nascent base pair is well stacked with the base pair at the primer-terminus. In the G*·G mismatch model, the incoming dGTP is syn and forms two hydrogen bonds with the anti -G* . However, the C1′–C1′ distance is enlarged to 11.9 ± 0.3 Å, and a somewhat less frequently sampled near reaction-ready Pα-O3′ distance is noted (68.1%).
|
16820532_p21
|
16820532
|
G·dNTP models
| 4.471251 |
biomedical
|
Study
|
[
0.9990431666374207,
0.0006433949456550181,
0.00031340779969468713
] |
[
0.9987255930900574,
0.0005979608977213502,
0.0004835706204175949,
0.0001927658449858427
] |
en
| 0.999998 |
Anti-G*·anti-dCTP major groove. With anti -G* opposite a Watson–Crick paired dCTP in the active site, the structural features are comparable to those of the unmodified control. In this anti -G*· anti -dCTP simulation, the Watson–Crick hydrogen bonds in the nascent base pair are highly occupied (>95% of the time); the C1′–C1′ distance has a normal average value of 10.9 ± 0.9 Å; the nascent base pair stacks well with the base pair at the primer-terminus, and the near reaction-ready Pα-O3′ distance is frequently sampled (82.4% of the time). However, two protein–DNA interactions have lower hydrogen bond occupancies in comparison to the unmodified control (Supplementary Table S6).
|
16820532_p22
|
16820532
|
G·dNTP models
| 4.368361 |
biomedical
|
Study
|
[
0.9991986155509949,
0.0005539436242543161,
0.0002474453067407012
] |
[
0.9991056323051453,
0.0004285339964553714,
0.0003196979232598096,
0.000146162899909541
] |
en
| 0.999999 |
Controls of anti-G·syn-dATP, anti-G·anti-dTTP and anti-G·syn-dGTP. These control models containing G·dATP, G·dTTP and G·dGTP mismatches without PhIP modification show distortions at the active sites in comparison to the Watson–Crick G·dCTP paired unmodified control ( Table 1 ). In the control of anti -G ·syn -dATP, although the C1′–C1′ distance has a close to normal value of 11.2 ± 0.2 Å (Supplementary Table S7), the full and the bifurcated hydrogen bonds in the nascent base pair have lower occupancies (<90% of the time, Supplementary Table S5), and the near reaction-ready Pα-O3′ distance is less frequently sampled (61.4% of the time, Supplementary Table S7). In the control of anti -G ·anti -dTTP, the C1′–C1′ distance has a normal value of 10.8 ± 0.2 Å (Supplementary Table S7). However, one of the two hydrogen bonds in the nascent pair is less frequently occupied (80.2% of the time, Supplementary Table S5), and the Pα-O3′ distance sampled the near reaction-ready range during only 59.7% of the time (Supplementary Table S5). In the control of anti -G ·syn -dGTP, the C1′–C1′ distance is enlarged to 11.9 ± 0.3 Å and one of the two hydrogen bonds in the nascent base pair is poorly occupied (only 46.6% of the time, Supplementary Table S5). However, the near reaction-ready Pα-O3′ distance is frequently sampled (76.7% of the time, Supplementary Table S5).
|
16820532_p23
|
16820532
|
G·dNTP models
| 4.362431 |
biomedical
|
Study
|
[
0.9991957545280457,
0.00047441967763006687,
0.00032983318669721484
] |
[
0.9992121458053589,
0.0003475838457234204,
0.00032849537092261016,
0.00011172089580213651
] |
en
| 0.999997 |
Anti-G*, syn-G* and unmodified control. In these models, the damaged template guanine has been skipped and the incoming dCTP pairs with the guanine on the 5′ side of the adduct . In comparison to the unmodified −1 control system, the two adduct systems, the anti -G* −1 deletion and syn -G* −1 deletion are significantly distorted. In the anti -G* −1 deletion model, the PhIP moiety is positioned on the major groove side, near-perpendicular to the template strand ; in the syn -G* −1 deletion model, the PhIP moiety is positioned on the minor groove side . Views of these ternary complexes and their active sites after 3 ns production MD simulations are shown in Supplementary Figures S3 and S4, and Figure 3 . For the syn -G* −1 deletion system, the PhIP rings are reoriented through rotation of α′ and β′ after about 320 ps of MD to avoid crowding between the distal phenyl ring and Lys78. This rearrangement results in a favorable environment around the PhIP ring system. The phenyl ring is in van der Waals contact with hydrophobic residue Ile104; the imidazo ring N3 and N4 atoms have favorable electrostatic interactions with the amino group of Lys78 (actual hydrogen bonds are formed ∼9% of the time). As shown in Supplementary Figure S8, the PhIP moiety is pocketed by the protein residues, the nascent base pair and the primer-terminus base pair.
|
16820532_p24
|
16820532
|
−1 Deletion models
| 4.401808 |
biomedical
|
Study
|
[
0.9991437196731567,
0.0006052693934179842,
0.0002509244077373296
] |
[
0.9989134073257446,
0.0005334771703928709,
0.0003668198187369853,
0.0001863739889813587
] |
en
| 0.999997 |
Watson–Crick hydrogen bonds in the nascent base pair are preserved in all three simulations (Supplementary Table S10). The C1′–C1′ distances are also normal in all cases (Supplementary Table S7), as is the chelation of the two Mg 2+ ions . Compared to the unmodified control, the primer end is repositioned towards the 5′ direction and away from the active site in the anti -G* and syn -G*adduct simulations ; this is more prominent in the syn -G* −1 deletion simulation, where the terminal primer base is shifted by about 3 Å. The backbone of the primer strand is concomitantly shifted, accompanied by small rotation of the thumb domain, which maintains contacts with the relocated backbone phosphate groups of the primer strand. In order to retain the B-DNA geometry, the backbone of the template strand is also adjusted, followed by small rotation of the little finger domain in order to maintain contacts with the same phosphate groups of the template strand. A similar relocation of the primer terminal base was observed in a Dpo4 crystal structure containing the 10 R (+)- cis-anti -[BP]- N 6 -dA adduct, with BP aromatic rings intercalated between the nascent and the primer terminal base pair ( 16 ). Here, the primer-terminus was positioned >10 Å away from the incoming dNTP. In both the anti -G* and syn -G* −1 deletion simulations, the dCTP loses its proper stacking contact with the ‘steric gate’ Tyr12 from the finger domain, and its hydrogen bonding with the finger residue Arg51. The separation of the dCTP from the finger domain could be a result of hydrogen bonds formed between the dCTP and the adduct (Supplementary Table S11). The Pα-O3′ distance is also affected by this primer side relocation. In the unmodified control system for the −1 deletion, the near reaction-ready Pα-O3′ distance is sampled during the whole simulation . In the anti -G* −1 deletion model, this distance is only achieved in the 380–480 ps time frame, and comes close to 4 Å occasionally after this time range . However, in the syn -G* −1 deletion case, the distance has an average value of 7.5 ± 0.7 Å throughout the simulation, and does not approach closer than 5.5 Å . In the syn -G* −1 deletion system, the stacking between the PhIP-modified syn -G and its 3′-neighbour is also disrupted . Thus, the −1 deletion simulations suggest various distortions likely to hinder nucleotide incorporation.
|
16820532_p25
|
16820532
|
−1 Deletion models
| 4.589175 |
biomedical
|
Study
|
[
0.9987074136734009,
0.0009398276451975107,
0.00035279805888421834
] |
[
0.9984235763549805,
0.0005323454970493913,
0.0007537908386439085,
0.00029032747261226177
] |
en
| 0.999999 |
Our simulations suggest that regardless of the incoming dNTP or whether the damaged base has a partner, the dG-C8-PhIP adduct is not likely to be accommodated in the minor groove side pocket of the Dpo4 DinB family polymerase ( Tables 1 and 2 ). When situated in this position, with the G* in the syn conformation, the bulky aromatic rings disrupt the geometry of the active site, particularly on the primer side . With the G* anti , however, the aromatic rings fit well on the major groove side. With dCTP opposite the anti -G*, Watson–Crick pairing is preserved and simulations show only modest distortions compared to the unmodified control (Supplementary Table S5 and Table 1 ). An incoming dTTP affords a well-formed wobble pair with only small distortions. In the case of the dATP, greater distortions are observed, with syn -dATP providing the more favorable structure, which contains hydrogen bonds employing the Hoogsteen edge of the dATP. Incoming dGTP was most distorted because even the syn conformation for dGTP caused enlargement of the nascent base pair. Furthermore, in the case of structures with no partner opposite the lesion, the −1 deletion models, the PhIP aromatic rings can again reside in the large major groove side open space; however, the active site region is notably distorted, and the approach of the primer-terminus to the α-phosphate of dNTP is inhibited by the PhIP moiety, particularly by its protruding methyl group .
|
16820532_p26
|
16820532
|
DISCUSSION
| 4.409895 |
biomedical
|
Study
|
[
0.9990899562835693,
0.0006538630113936961,
0.0002561866131145507
] |
[
0.999015212059021,
0.000341674400260672,
0.0004732770612463355,
0.0001698026026133448
] |
en
| 0.999996 |
In contrast, an earlier study of the 10 S (+)- trans-anti -[BP]- N 2 -dG showed that this adduct can be accommodated reasonably well on the minor groove side of the Dpo4, irrespective of incoming dNTP, with only modest enzyme perturbation including opening of the little finger and some small rearrangement of active site region residues ( 54 ). In this case, the modified dG adopts the anti conformation. The major groove position with the N 2 -dG adduct in syn conformation also provides a good accommodation for the BP ring system. Experimental kinetic studies revealed promiscuous nucleotide incorporation for this case. Running-start primer extension experiments indicated that the damage can be bypassed to a significant extent ( 54 ).
|
16820532_p27
|
16820532
|
DISCUSSION
| 4.315658 |
biomedical
|
Study
|
[
0.9993221759796143,
0.00036955156247131526,
0.0003082849725615233
] |
[
0.9992550015449524,
0.0003148200339637697,
0.0003371515776962042,
0.00009305325511377305
] |
en
| 0.999997 |
Figure 4 illustrates the differential accommodation of the C8 and N 2 -dG adducts in the Dpo4 minor groove, showing why the C8 adduct is not well positioned there, while the N 2 adduct is, regardless of dNTP . Specifically, this N 2 adduct ring system is directed 5′ along the template strand in our models; however, the C8 adduct rings are oriented 3′ along the template strand, protrude to the primer side and thereby disrupt the active site directly where the nucleotidyl transfer reaction is to take place. Perhaps C8-dG bulky adducts, such as dG-C8-PhIP and dG-C8-AAF might less readily allow nucleotide incorporation by Dpo4, in part because of their poor accommodation in the enzyme minor groove side pocket; thus, only one site is available to harbor the lesion, while N 2 adducts, such as 10 S (+)- trans-anti -[BP]- N 2 -dG can reside in either major or minor groove side pockets.
|
16820532_p28
|
16820532
|
DISCUSSION
| 4.411412 |
biomedical
|
Study
|
[
0.9993940591812134,
0.000374653929611668,
0.00023124214203562587
] |
[
0.9983800649642944,
0.0008401808445341885,
0.0006437385454773903,
0.00013603460683953017
] |
en
| 0.999997 |
Additionally, our structures suggest that translocation may be difficult for such major groove positioned multi-ringed adducts. Specifically, a recent crystal structure of Dpo4 has suggested that translocation starts with movement of the little finger during nucleotide binding, followed by thumb movement during the chemical reaction ( 23 ). The replication cycle involves a screw-like counterclockwise rotation/translocation of the polymerase along the DNA helix axis (viewed in the 5′ to 3′ direction of the template strand). This threading of the DNA through the polymerase places the next templating base in the active site, awaiting entry of its complementary dNTP. Prior study of the dG-C8-AAF adduct suggested that the preferred 3′-directed orientation of the fluorenyl ring system along the template strand, in the major groove, would impede the rotation of the little finger ( 34 ); this impediment is not due to the acetyl group. The current study of the dG-C8-PhIP adduct suggests a similar impact on translocation. As shown in Figure 5a and b , in the insertion position the adduct in the anti conformation is enveloped by the little finger without collision . However, once in the post-insertion site, the adduct would be in collision with the little finger, indicating that translocation to the post-insertion site would be difficult. Very large rearrangement of the PhIP adduct would be required to move it to another orientation that allows translocation. Additional bypass polymerases may be required to further extend beyond the lesion ( 4 ). Furthermore, it appears that N 2 adducts, such as 10 S (+)- trans-anti -[BP]- N 2 -dG, would be less likely to impede translocation by colliding with the little finger, when in the anti conformation and placed in the minor groove side polymerase pocket . The number of rings in the adduct together with their orientation will determine the extent of these effects on translocation and this remains to be elucidated.
|
16820532_p29
|
16820532
|
DISCUSSION
| 4.550587 |
biomedical
|
Study
|
[
0.998890221118927,
0.0007549886940978467,
0.0003548082895576954
] |
[
0.9984706044197083,
0.0005831218441016972,
0.0007043996010906994,
0.00024187345115933567
] |
en
| 0.999997 |
Primer extension studies of dG- N 2 -AAF and dG-C8-AAF with hpol κ have shown that the frequency of bypass of dG- N 2 -AAF is at least 4 orders of magnitude higher than for dG-C8-AAF, and it was proposed that the observed difference may be due to the different linkage site ( 70 ). This is consistent with our suggested poorer accommodation on the minor groove side of C8 than N 2 adducts. However, we do not know exactly how structurally similar pol κ and Dpo4 ternary complexes are, since to date, only a crystal structure of the apo-pol κ catalytic core is available ( 71 ).
|
16820532_p30
|
16820532
|
DISCUSSION
| 4.223112 |
biomedical
|
Study
|
[
0.9994656443595886,
0.0002491424384061247,
0.0002852653560694307
] |
[
0.9992513060569763,
0.00024439796106889844,
0.00043794058728963137,
0.00006640850187977776
] |
en
| 0.999998 |
Furthermore, our results show that the PhIP-modified lesion produces less distorted structures for G*·dTTP and G*·dATP ( anti -G* ·anti -dTTP and anti -G* ·syn -dATP) than for their respective unmodified controls ( anti -G ·anti -dTTP and anti -G ·syn -dATP) ( Table 1 ). The structural origin of this interesting phenomenon is at least partly in the formation of hydrogen bonds between the PhIP rings and the little finger residue Arg332, which stabilize the anti conformation in the adduct . These findings would be relevant to observed mutagenic behavior of PhIP in inducing G to T transversions and G to A transitions in mammalian systems ( 44 – 52 ), if error-prone incorporation opposite the lesion involved the human DinB polymerase pol κ, and its structural properties prove similar to those of its prokaryotic homolog Dpo4.
|
16820532_p31
|
16820532
|
DISCUSSION
| 4.277998 |
biomedical
|
Study
|
[
0.9994125366210938,
0.00034589317510835826,
0.00024160525936167687
] |
[
0.9993801116943359,
0.00023862271336838603,
0.00029434767202474177,
0.00008681141480337828
] |
en
| 0.999997 |
In conclusion, our modeling and MD simulations for dG-C8-PhIP suggest that the adduct would increase the infidelity of Dpo4 and hinder translocation by the enzyme. We hope that the hypotheses resulting from our modeling studies will provide useful suggestions for future experimental investigations.
|
16820532_p32
|
16820532
|
DISCUSSION
| 4.085067 |
biomedical
|
Study
|
[
0.9993714690208435,
0.0001642194256419316,
0.00046439794823527336
] |
[
0.9981189370155334,
0.0013483616057783365,
0.00042440200923010707,
0.00010820692841662094
] |
en
| 0.999997 |
Supplementary Data are available at NAR online.
|
16820532_p33
|
16820532
|
SUPPLEMENTARY DATA
| 0.985075 |
biomedical
|
Other
|
[
0.7697952389717102,
0.0054101841524243355,
0.22479452192783356
] |
[
0.01419480424374342,
0.982682466506958,
0.0018953380640596151,
0.00122743786778301
] |
en
| 0.999995 |
It is well established that DNA adopts various conformations and all alternative forms of DNA are restricted to a small subset of nucleotide sequences ( 1 , 2 ). The human genome, like other mammalian genomes has very high proportion of repeated DNA sequences ( 3 ). Other than the direct repeats within the non-coding and occasionally within coding regions, there are defined ordered sequences that contain various symmetry elements viz. inverted repeats (palindromes) and mirror repeats. The inverted repeat sequences that are not completely symmetrical or that have a center region which is not an inverted repeat is called imperfect palindrome or quasipalindromes. Because of their nature, inverted repeats in DNA and RNA can engage in intra- and intermolecular base pairing forming a variety of structural forms like hairpins, bulges, internal loops, cruciforms, Holliday junctions etc ( 4 – 6 ).
|
16855288_p0
|
16855288
|
INTRODUCTION
| 4.28744 |
biomedical
|
Study
|
[
0.9994333386421204,
0.00017204516916535795,
0.00039461159030906856
] |
[
0.9390853643417358,
0.004982516635209322,
0.05571044608950615,
0.00022168223222251981
] |
en
| 0.999996 |
The biological relevance of palindromic and quasipalindromic sequences is clear from their occurrence at functional recognition sites in DNA ( 7 , 8 ). Eukaryotic DNA, in contrast to that of prokaryotes is characterized by having large palindromic regions. Often inverted repeats occur near putative control regions of genes or at origin of DNA replication ( 9 ). In the linear form they play important biological roles as binding sites (operator sequences) for dimeric proteins (repressors and activators) ( 8 ). Owing to their distinct structure, hairpins may offer binding sites for proteins ( 10 , 11 ). Role of DNA secondary structure in the initiation of viral DNA replication has been reported ( 12 ). Experiments showed that one of the strands in the double-stranded molecule at its palindromic stretches adopts a hairpin with AT rich loop and is specially recognized by the origin binding protein. DNA hairpins and cruciforms are determinants for topoisomerase II recognition and cleavage ( 13 , 14 ).
|
16855288_p1
|
16855288
|
INTRODUCTION
| 4.503199 |
biomedical
|
Study
|
[
0.9993089437484741,
0.0003265168925281614,
0.0003645214019343257
] |
[
0.9871980547904968,
0.0005128448829054832,
0.01213447842746973,
0.00015471006918232888
] |
en
| 0.999997 |
Imperfect inverted repeats undergo spontaneous mutation to more-perfect inverted repeats and this correction is a general mechanism for mutation in prokaryotes. This was confirmed by an analysis, where relative frequency of quasipalindromes and perfect palindromes in more than 100 sequenced prokaryotic genomes was determined and found that perfect palindromes were relatively more frequent than quasipalindromes ( 15 ). Mutational hotspots in natural quasipalindrome in Escherichia coli are recently been reported ( 16 , 17 ). The ability to adopt hairpin and cruciform secondary structures by imperfect inverted repeats is associated with frameshift mutations. Several human genetic diseases illustrate inverted repeat mediated mutagenesis ( 18 ). The role of hairpin formation in Fredreich's ataxia triplet repeat expansion has recently been reported ( 19 ). A recent elegant survey describes some principles for the formation of unusual DNA duplex and hairpin motifs ( 20 ).
|
16855288_p2
|
16855288
|
INTRODUCTION
| 4.437931 |
biomedical
|
Study
|
[
0.9992538094520569,
0.0003333209897391498,
0.00041293047252111137
] |
[
0.9526471495628357,
0.0007055571768432856,
0.046401601284742355,
0.0002456242509651929
] |
en
| 0.999998 |
Locus control regions (LCRs), first defined in the human β-globin locus are operationally defined by their ability to enhance the expression of linked genes to physiological level in a tissue-specific and copy number-dependent manner at ectopic chromatin sites ( 21 , 22 ). The presence of homeodomain protein binding sites, inverted repeats and nuclear matrix attachment regions along the β-globin gene cluster is well documented ( 23 ). Dyad symmetry sequences are present close to the four DNase I hypersensitive site (HS) in the β-globin LCR. More recent studies ( 24 ), on the spectrum of β-thalassemia mutation and their association with allelic sequence polymorphism at the human β-globin gene cluster have revealed a single nucleotide polymorphism (SNP) at the quasipalindromic sequence of HS4 of the LCR. The studies carried out on Indian population showed an A → G polymorphism in the sequence d-TGGGG(A/G)CCCCA ( 24 ). A significant association was observed between the G allele and the occurrence of β-thalassemia. It was concluded that it was an evolutionarily new mutation and was hypothesized that the quasipalindromic stretch might exist in a hairpin form, where the A/G might form single residue loop.
|
16855288_p3
|
16855288
|
INTRODUCTION
| 4.427084 |
biomedical
|
Study
|
[
0.9994840621948242,
0.00026868865825235844,
0.0002472786291036755
] |
[
0.9986022114753723,
0.00024044298334047198,
0.0010601282119750977,
0.00009729516750667244
] |
en
| 0.999996 |
Our recent study ( 25 ) which was an attempt to work out the said hypothesis, reported the structure of the quasipalindromic sequence TGGGG(A/G)CCCCA displaying A → G SNP. Interestingly, the multiple sequence alignment of HS4 region in β-globin gene cluster from different organisms (rabbit, mouse, bovine, galago and goat) revealed that the quasipalindromic stretch studied here is unique to Homo sapiens . This imperfect palindrome exhibited a hairpin-duplex equilibrium at near physiological solution conditions. Dependence of CD spectra on oligomer concentration manifested in the shift of CD signals from 265 to 285 nm position was interpreted in terms of interconversion of A → B form of DNA, where hairpin species adopts A-form, while B-form is the preferred conformational state of intermolecular duplex.
|
16855288_p4
|
16855288
|
INTRODUCTION
| 4.231703 |
biomedical
|
Study
|
[
0.9995126724243164,
0.00022856520081404597,
0.0002587758644949645
] |
[
0.999427080154419,
0.00020915918867103755,
0.0003040886658709496,
0.00005976222746539861
] |
en
| 0.999997 |
Among the polymorphic conformations, the structural elements viz. hairpins, bulges, internal loops may be considered important fundamental building blocks in nucleic acid structure. Structural transitions between various forms of DNA would have consequences in vivo , and a thorough understanding of their physical and structural properties is consequential. With these concepts in mind and prompted by our previous study ( 25 ) and by others work on similar lines ( 26 – 29 ), we undertook structural study on the perfect duplexes [HP(A+T)11/HP(C+G)11] of 11 nt quasipalindrome 5′-TGGGG(A/T/C/G)CCCCA-3′ namely, (HPA11/HPT11/HPC11/HPG11) and the perfect duplexes of its extended versions [HP(A+T)21/HP(C+G)21], the flanked 21 nt long sequences [5′-GCTCTTGGGG(A/G)CCCCAGTACA-3′ namely, (HPA21/HPG21)]/[5′-TGTACTGGGG(T/C)CCCCAAGAGC-3′namely (HPT21/HPC21)] presentin the HS4 of major regulatory LCR of β-globin gene. Using gel-electrophoresis, ultraviolet (UV)-thermal denaturation, circular dichroism (CD) techniques, we demonstrate the structural transitions within a perfect duplex to hairpin and bulge duplex/cruciforms. The characterization of such polymorphic sites at palindromic/quasipalindromic sequences is of crucial importance in understanding the biological functions of DNA. Unusual structural motifs may also represent novel targets for pharmaceutical research.
|
16855288_p5
|
16855288
|
INTRODUCTION
| 4.332349 |
biomedical
|
Study
|
[
0.999497652053833,
0.0003017786657437682,
0.00020056682114955038
] |
[
0.9987448453903198,
0.0002823190006893128,
0.0008751266286708415,
0.00009775916987564415
] |
en
| 0.999996 |
The oligonucleotides, synthesized in 1 μM scale by Bio Basic Inc., Canada, were received as PAGE purified in the form of lyophilized powder. They were stored at −20°C and were used without further purification. The concentration of the oligonucleotides was determined spectrophotometrically by using the extinction coefficient (ɛ) calculated by nearest neighbour method ( 30 ) and measuring the absorbance at 260 nm at elevated temperature (95°C), following the method described earlier ( 31 ). The ɛ values used for 11mer oligo-sequences d-TGGGGACCCCA (HPA11), d-TGGGGGCCCCA (HPG11), d-TGGGGTCCCCA (HPT11) and d-TGGGGCCCCCA (HPC11) were 104 200, 100 900, 100 700 and 98 000 M −1 cm −1 , respectively and for 21mer oligonucleotides d-GCTCTTGGGG A CCCCAGTACA (HPA21), d-GCTCTTGGGG G CCCCAGTACA (HPG21), d-TGTACTGGGG T CCCCAAGAGC-3′ (HPT21) and d-TGTACTGGGG C CCCCAAGAGC-3′ (HPC21) were 196 700, 193 400, 198 800 and 196 100 M −1 cm −1 , respectively. d-TAAAAAT (SC, ɛ = 78 800 M −1 cm −1 ), d-CTTGAGCTCAAG (PAL12, ɛ = 113 700 M −1 cm −1 ), d-CGCGCGCGCGCGCGCGCGCG (PAL20, ɛ = 168 300 M −1 cm −1 ), were used as size markers in gel assays.
|
16855288_p6
|
16855288
|
MATERIALS AND METHODS
| 4.134386 |
biomedical
|
Study
|
[
0.9995504021644592,
0.00019023529603146017,
0.0002592937962617725
] |
[
0.9992280006408691,
0.00041856247116811574,
0.0003002133744303137,
0.000053208765166345984
] |
en
| 0.999994 |
For simpler understanding, the duplexes formed by mixing equimolar concentration of studied oligonucleotides will be named as HP(A+T)11, HP(C+G)11, HP(A+T)21 and HP(C+G)21.
|
16855288_p7
|
16855288
|
MATERIALS AND METHODS
| 3.352582 |
biomedical
|
Study
|
[
0.9967002272605896,
0.000259863561950624,
0.0030398780945688486
] |
[
0.8185888528823853,
0.178226500749588,
0.0027447338216006756,
0.0004399483441375196
] |
en
| 0.999996 |
The stock solutions of the oligomers were prepared by dissolving directly the lyophilized powder in MilliQ water. All other chemicals were of analytical grade. The buffer solution consisted of 20 mM sodium cacodylate (pH 7.4), 0.1 mM EDTA containing 100 mM NaCl.
|
16855288_p8
|
16855288
|
MATERIALS AND METHODS
| 3.535512 |
biomedical
|
Study
|
[
0.9991496801376343,
0.00022391641687136143,
0.0006263944669626653
] |
[
0.9230676293373108,
0.07577157765626907,
0.0007172309560701251,
0.0004435605078469962
] |
en
| 0.999998 |
For performing gel assays, oligonucleotide samples were prepared in 20 mM sodium cacodylate buffer (pH 7.4) at desired concentrations. The final volume of the sample in the buffer was 20 μl. Importantly prior to performing gel assays in non-denaturating conditions, the purity of the commercially made oligomers was checked by running them on 20% PAGE using 7 M urea. They migrated as single bands. For non-denaturating gel assays, the samples (20 μl) of total volume, were heat treated at 95°C for 5 min and slowly cooled to room temperature over about 10 h. The oligonucleotides (at 10 μM strand concentrations) samples were incubated at 4°C for 3 h before loading onto 10% polyacrylamide gel pre-equilibrated for 2 h. The gel contained 20 mM sodium cacodylate (pH 7.4) with 100 mM NaCl and 0.1 mM EDTA and the running buffer being 1× TBE with 100 mM NaCl. For simplicity, the salt is designated as Na + cations at appropriate places in the text. Tracking dye consisted of Orange-G. The gels were run at a constant voltage of 40 V at room temperature (25°C). After electrophoresis the gels were stained with Stains-all (Sigma) solution and finally visualized under white light and photographed by Alphalmager™ 2200 (Alpha Infotech Corp.). We would like to add here that in our previous report ( 25 ), gel experiments were performed using radiolabelling method for detecting oligomer structures. Interestingly, we were able to get identical results using staining (stains-all/silver staining) method. However, we found that use of stains did not change the concept of our results.
|
16855288_p9
|
16855288
|
Non-denaturating gel-electrophoresis
| 4.138486 |
biomedical
|
Study
|
[
0.9995793700218201,
0.00021111023670528084,
0.00020945616415701807
] |
[
0.9989263415336609,
0.0005497701349668205,
0.0004596724174916744,
0.00006415566895157099
] |
en
| 0.999997 |
The thermal denaturation experiments were performed on a Varian make CARY-100 Spectrophotometer equipped with a peltier thermo-programmer and interfaced with aPentium III computer for data collection and analysis. The stoppered quartz cuvettes of 10 and 1 mm optical path length with 1 and 0.35 ml volume, respectively, were used for the measurements. The oligonucleotide samples were prepared by taking their appropriate range of strands concentrations, heating the samples up to 100°C for 5 min followed by slow cooling. The temperature dependence on the absorption value of the DNA was monitored at 260 nm. The temperature of the cell holder was increased from 0 to 100°C at a rate of 0.5°C/min. A teflon-coated temperature probe, immersed directly in a control cuvette, measured the sample temperature. The sample solutions were overlaid with paraffin-oil to prevent evaporation. The melting curves were normalized at lower/higher temperature values. The thermal melting temperature ( T m ) was determined from the peak of the computer generated first derivative of the absorbance verses temperature profile. The accuracy of the reported T m values is ±1°C
|
16855288_p10
|
16855288
|
UV-thermal denaturation
| 4.230238 |
biomedical
|
Study
|
[
0.9994297623634338,
0.0003527775697875768,
0.00021739884687121958
] |
[
0.9988777041435242,
0.00045143309398554265,
0.0005809121648781002,
0.00008990417700260878
] |
en
| 0.999998 |
For secondary structure determination, CD Spectra were recorded on JASCO-715 Spectropolarimeter interfaced with an IBM PC compatible computer, calibrated with D-Camphor Sulphonic acid. Five scans of the spectrum were collected over the wavelength range of 220–320 nm at a scanning rate of 100 nm/min. The average of multiple scans was used for analysis. The scan of the buffer alone recorded at room temperature was subtracted from the average scans for each DNA duplex. Data were collected in units of millidegrees versus wavelength and normalized to total DNA concentration.
|
16855288_p11
|
16855288
|
Circular dichroism
| 4.16515 |
biomedical
|
Study
|
[
0.9995642304420471,
0.00025852699764072895,
0.00017723451310303062
] |
[
0.9989822506904602,
0.0005186270573176444,
0.00042135443072766066,
0.0000777325767558068
] |
en
| 0.999995 |
Non-denaturating gel studies reported previously ( 25 ), revealed that the individual strands HPA11, HPG11 and the extended version of HPG11 (the flanked HPG21), do exist as unimolecular and bimolecular species corresponding to hairpin and bulge duplex conformations, respectively. Further, in this report their duplex forming ability was investigated in presence of their complementary strands (HPT11, HPC11 and flanked HPC21), HPA21 and HPT21. The duplex was prepared in 20 mM sodium cacodylate buffer (pH 7.4) containing 100 mM NaCl and 0.1 mM EDTA by mixing equimolar concentrations of 11mer as well as 21mer oligonucleotides with their counterparts at elevated temperature, followed by slow cooling. Such duplex samples were run on a non-denaturating gel at room temperature (∼25°C). The gel was run under constant voltage until the desired separation was achieved. The electrophoretogram of the two duplexes under study is shown in Figure 1a and b .
|
16855288_p12
|
16855288
|
Non-denaturating gel-electrophoresis
| 4.138145 |
biomedical
|
Study
|
[
0.9994124174118042,
0.0002487706660758704,
0.00033883709693327546
] |
[
0.9995205402374268,
0.00019160717783961445,
0.0002386518899584189,
0.0000492315593874082
] |
en
| 0.999996 |
To our surprise, the gel mobility pattern obtained for various mixtures of oligomers, did not differ much from the ones obtained with individual strands under identical conditions ( 25 ). The duplexes HP(C+G)11 and HP(C+G)21 at 10 μM strand concentration and even the individual HPC11, HPC21 (lanes 1 and 3) and HPT21, HPT11 strands (lanes 8 and 9) exhibited two distinct bands. To predict the molecularity of the structural species formed by the oligomers under study, three control strands, a heptamer marker (SC; lanes 5 and 11), a 12 bp (PAL12; lanes 6 and 10) and 20 bp (PAL20; lane 7) palindromic sequences, are used as size markers. In Figure 1 , the lower band of HPC11 (lane 1) and HPT11 (lane 9) migrate equivalent to the SC marker (lane 5) indicating migration of HPC11 and HPT11 as folded species (hairpin form), as has been shown for HPG11 and HPA11 ( 25 ), while the upper band of HPC11 migrates corresponding to PAL12 (lane 6) and can be assumed a 11 bp bulge duplex. It signifies that individual strands of the short 11mer perfect duplexes are capable of existing in hairpin and bulge duplex forms, independently. Thus it seems conceivable to assume that of the two bands appeared in case of duplex HP(C+G)11 sample (lane 2), the lower band corresponds to the folded (hairpin) form of individual strands, while the bulge duplex and perfect duplex species having identical size, will occupy the slow moving upper band. The possible structures of hairpin and bulge duplex forms are depicted in Figure 5a.
|
16855288_p13
|
16855288
|
Non-denaturating gel-electrophoresis
| 4.259472 |
biomedical
|
Study
|
[
0.99919193983078,
0.0003365936572663486,
0.00047144590644165874
] |
[
0.9994056224822998,
0.00023293674166779965,
0.00029933781479485333,
0.00006205592217156664
] |
en
| 0.999998 |
Similarly, of the two bands exhibited by HPC21 and HPT21 where the core HPC11 sequence was flanked by naturally occurring 5 nt non-complementary stretch on both the 5′- and 3′-sides, the lower band moving equivalent to PAL12 corresponds to the folded form of 21mer oligonucleotide, as the hairpin form of HPC21 and HPT21 with unpaired flanking ends. Electrophoretic mobility of single-stranded DNA becomes almost the same as that of half-length double-stranded DNA when it makes a hairpin structure ( 26 ) and hairpins migrate faster than their corresponding unstructured single-strands ( 32 , 33 ). The upper slow migrating band of HPC21 with equivalent mobility to the 20 bp palindromic duplex (PAL20, lane 7), is interpreted in terms of a bulge duplex species of HPC21 and HPT21 containing the unpaired flanking sequences . It seems reasonable to mention here that an identical behavior was displayed by the HPG21 strand, whose highly retarded upper band was regarded due to an extended bulge/cruciform like structure. Based on the observed status of HPG21 studied previously ( 25 ) and of HP(C+G)21 in the present study under similar experimental conditions, one can anticipate that the inherent tendency of forming stable secondary structures by each complementary strand may interfere in the formation of the perfect duplexes. Interestingly, such an assumption is reflected in the gel pattern, where a 1:1 mixture HP(C+G)21, of the two complementary strands i.e. HPG21 and HPC21 exhibited two bands , the fast moving lower band corresponds to the hairpin form of the HPC21 and HPG21 with unpaired flanking ends and the slow moving upper band possibly might correspond to the perfect duplex or a compact cruciform structure containing two hairpins on each G and C strands . Interestingly the gel mobility patterns similar to HPC21 discussed above, was also displayed by HPT21 which further confirms the formation of two structural species by this strand. Thus it can be concluded that the component strands of the short 11mer and long 21mer perfect duplexes, are capable of existing in hairpin and bulge duplex forms, independently .
|
16855288_p14
|
16855288
|
Non-denaturating gel-electrophoresis
| 4.503078 |
biomedical
|
Study
|
[
0.9991330504417419,
0.0004264571180101484,
0.0004405523359309882
] |
[
0.998918890953064,
0.00037182142841629684,
0.0005971091450192034,
0.00011226508649997413
] |
en
| 0.999997 |
Worth mentioning is the difference observed in the mobility of upper bands of duplex HP(C+G)21 and individual strand HPC21 (lane 3). The bulge duplex or cruciform like structure formed by HPC21 oligonucleotide has two non-complementary 5 nt flanking ends, while the possible cruciform structure generated in the duplex [HP(C+G)21] contains two hairpins and paired flanking duplex region, producing overall a compact cruciform structure . Apparently this compact form of [HP(C+G)21] moves faster than the bulge duplex form of HPC21, giving a different mobility pattern in the gel. Importantly at this stage, the possibility of formation of perfect linear duplex due to Watson–Crick pairing of HPC21 and HPG21 strands cannot be ruled out.
|
16855288_p15
|
16855288
|
Non-denaturating gel-electrophoresis
| 4.229933 |
biomedical
|
Study
|
[
0.9993793964385986,
0.0002378067292738706,
0.00038281051092781126
] |
[
0.9992448091506958,
0.00046754334471188486,
0.00022911973064765334,
0.000058572062698658556
] |
en
| 0.999998 |
The native PAGE experiments were also run in cold room (8–10°C). Except a longer electrophoresis run time, no difference was found with the results of electrophoretic mobility pattern of the same oligomers, run at room temperature (∼25°C).
|
16855288_p16
|
16855288
|
Non-denaturating gel-electrophoresis
| 2.922486 |
biomedical
|
Study
|
[
0.9949860572814941,
0.00043888739310204983,
0.004575030878186226
] |
[
0.9869332313537598,
0.01194778736680746,
0.0009055890841409564,
0.00021337586804293096
] |
en
| 0.999998 |
More than a decade ago, McMurray's study ( 27 ) on human enkephalin gene has demonstrated reversible conformational transition from a 23 bp duplex containing the enhancer, to two individual hairpin structures, each formed from one strand of the duplex. A structural model was suggested describing that DNA secondary structure within the enhancer region plays an active role in cAMP-inducible activation via the formation of cruciform structures. Later study ( 34 ) using structural methods concluded that each oligonucleotide strand exists primarily as a hairpin structure over a wide range of oligomer concentration and temperature. Role of cruciform structure in transcription regulation of enkephalin gene was discussed.
|
16855288_p17
|
16855288
|
Non-denaturating gel-electrophoresis
| 4.262028 |
biomedical
|
Study
|
[
0.9995156526565552,
0.00021154257410671562,
0.000272915989626199
] |
[
0.984782874584198,
0.0004792980616912246,
0.014588887803256512,
0.00014889103476889431
] |
en
| 0.999997 |
Reports on structural polymorphism including duplex–tetraplex equilibrium ( 35 , 36 ), structural competition between the G-quadruplex, i-motif and Watson–Crick duplex ( 37 ) again indicate the importance of elucidation how alternative DNA structures with biological implications form.
|
16855288_p18
|
16855288
|
Non-denaturating gel-electrophoresis
| 3.976719 |
biomedical
|
Study
|
[
0.9992691874504089,
0.00013492674042936414,
0.000595819961745292
] |
[
0.9563637375831604,
0.01287722960114479,
0.030537908896803856,
0.00022108318808022887
] |
en
| 0.999999 |
In the following section a correlation between the gel studies and UV-thermal denaturation profiles further establishes the presence of various structural possibilities of the studied sequences.
|
16855288_p19
|
16855288
|
Non-denaturating gel-electrophoresis
| 2.909914 |
biomedical
|
Study
|
[
0.9884059429168701,
0.000617783167399466,
0.01097632385790348
] |
[
0.9717845916748047,
0.02475830540060997,
0.003130352823063731,
0.00032674067188054323
] |
en
| 0.999999 |
The absorbance versus temperature melting profile shown in Figure 2a for the HP(C+G)11 duplex at 1 μM concentration, containing equimolar quantities of HPC11 and its counter part 11mer, HPG11 did not display the characteristic monophasic sigmoidal curve, generally expected for a perfect duplex. Surprisingly, a triphasic melting profile with three transitions was obtained. The upper transition could not attain a proper plateau. The derivative plot of the melting profile shown as inset shows a major peak depicting a T m of 58°C for the middle transition, while the T m for upper transition was ∼90°C. A small hump on the derivative plot centered around 43°C cannot be ignored as it seem to be originated from the lower portion of the melting profile. The occurrence of triphasic melting profiles can be interpreted as presence of more than one structural species in the solution. Thus it can be assumed that equimolar mixture of HPC11 and HPG11 also generated structures, other than a perfect duplex species. The interpretation of UV-melting profile present in Figure 2a can be simplified by considering the status of individual quasipalindromic strands. In our previous study ( 25 ), HPG11 has been shown to exist in hairpin-duplex equilibrium under identical solution conditions used in present study. Thus the lower, middle and higher temperature transitions correspond to the melting of bulge duplex, perfect duplex and hairpin forms, respectively.
|
16855288_p20
|
16855288
|
UV-thermal denaturation experiments
| 4.274639 |
biomedical
|
Study
|
[
0.9993157386779785,
0.0003099837340414524,
0.0003742863773368299
] |
[
0.9994624257087708,
0.0001835764414863661,
0.0002934307267423719,
0.00006049420335330069
] |
en
| 0.999998 |
Further, to study the effect of the flanking sequences on the hybridization capabilities of complementary quasipalindromic sequence, thermal denaturation experiments were performed on HP(C+G)21, containing equimolar concentrations of complementary HPC21 and HPG21 strands. HPC21/HPG21 are the 5′- and 3′- 5 nt extensions of HPC11/HPG11. To our surprise, the UV-melting profile of HP(C+G)21 (2 μM) shown in Figure 2b , also displayed a pattern, moderately identical to the melting of HP(C+G)11. The two transitions corresponding to two peaks in the derivative plot (inset) indicated the presence of two structural species and can be interpreted on somewhat similar logics, as was made for HP(C+G)11. On that account, since the sequence HPG21 under identical solution conditions has already been shown to exist in equilibrium with hairpin and bulge duplex forms indicating T m values of >90 and 40°C for the two forms, respectively ( 25 ), the higher temperature transition ( T m ∼ 90°C) is consistent with the melting of intramolecular hairpin forms of individual HPG21 and HPC21 strands. The lower temperature transition showing higher hyperchromicity than the upper transition, might correspond to the melting of intermolecular bulge duplexes formed by HPC21 and duplexes formed by perfect pairing of HPC21 with HPG21. However, possibilities of other structures cannot be ruled out at this stage.
|
16855288_p21
|
16855288
|
UV-thermal denaturation experiments
| 4.263202 |
biomedical
|
Study
|
[
0.9993413090705872,
0.00034373774542473257,
0.00031502023921348155
] |
[
0.9994394183158875,
0.000201483751880005,
0.0002872950863093138,
0.00007179884414654225
] |
en
| 0.999996 |
It is important to mention that the said species could also include the structures generated by association of two hairpin conformers via Watson–Crick base pairing between the flanking nucleotides of complementary strands, finally shaping into cruciforms. Compared to bulge duplexes (with flanking ends) formed by individual HPG21 and HPC21 strands, cruciform structures [formed by HP(C+G)21], which engage both the complementary strands, are compact structures . Though the calculated T m of 67°C for melting of discrete structures manifested in single broad melting profile, cannot be assigned definitively to one structural form. The differential gel pattern shown by HPC21 and HP(C+G)21 however, reflects in the possibility of compact cruciform structure formed by HPC21 and HPG21 strands. This observation gave us a clue to rationalize that the lower temperature broad transition ( T m = 67°C) in the melting curve of HP(C+G)21 most likely corresponds to the disordering of cruciform species. The possibility of involvement of a cruciform intermediate in hairpin-duplex interconversion via a cruciform intermediate has been well suggested ( 26 , 38 ).
|
16855288_p22
|
16855288
|
UV-thermal denaturation experiments
| 4.491241 |
biomedical
|
Study
|
[
0.9992029070854187,
0.0004015535523649305,
0.0003956214932259172
] |
[
0.9988334774971008,
0.00045102235162630677,
0.0005926985177211463,
0.00012272650201339275
] |
en
| 0.999996 |
Considering the overall interpretation of the thermal melting profiles of HP(C+G)11 and HP(C+G)21 , it can very well be concluded that hybridization of DNA oligonucleotide to their complementary counterparts is complicated by the presence of secondary structures. Presence of palindromic region though intervened by single base in HPG11/HPG21 and HPC11/HPC21 can still facilitate the formation of intra- and intermolecular Watson–Crick base pairing between the complementary ends resulting into hairpin/bulge duplex/bulge duplex with flanking ends or cruciform structures.
|
16855288_p23
|
16855288
|
UV-thermal denaturation experiments
| 4.299049 |
biomedical
|
Study
|
[
0.9995452761650085,
0.0002077223762171343,
0.00024704250972718
] |
[
0.9976016879081726,
0.0017150260973721743,
0.0005776018369942904,
0.00010569319420028478
] |
en
| 0.999998 |
For the information on the molecularity of both the structural species detected in native PAGE, a dependence of oligomer concentration of HPG21 sequence on the T m was carried out at 100 mM NaCl concentration. The melting profiles along with their derivatives (inset) shown in Figure 3 , are distinctly biphasic at oligomer concentrations from 10 to 40 μM, again suggesting melting of the two ordered forms. Since both, the lower and higher temperature transitions of the biphasic curves were sufficiently separated, it was possible to extract two T m 's for two ordered forms. The actual T m 's were determined from the first derivative of the observed thermal transition. The biphasic curve obtained for HPG21 at 10 μM concentration corresponds to the lower temperature ( T m 50°C) and higher temperature transition ( T m 93°C), whereas at 40 μM concentration, the oligonucleotide exhibited melting at 57 and 93°C for the lower and higher transitions, respectively. As expected for bimolecular (intermolecularly folded) structures adopted by HPG21, its first transition showed oligomer concentration dependence on T m , whereas the second thermal transition remains relatively independent of oligomer concentration, revealing the presence of monomolecular (intramolecular duplex) species. It concludes that like HPG11, the oligomer HPG21 also exists in hairpin-duplex equilibria, and it is due to this property that its hybridization with HPC21 is hindered.
|
16855288_p24
|
16855288
|
UV-thermal denaturation experiments
| 4.424526 |
biomedical
|
Study
|
[
0.9992856383323669,
0.0004265539755579084,
0.00028781796572729945
] |
[
0.9991428852081299,
0.00028114402084611356,
0.0004661224957089871,
0.00010990173177560791
] |
en
| 0.999997 |
More than a decade ago Hirao et al. ( 39 , 40 ) in a study on short (heptamers and octamers) DNA fragments reported the formation of extraordinarily stable mini-hairpin structures containing one or three residue loops with the melting temperature as high as 76°C in 100 mM NaCl. The origin of such an unusual stability was revealed by solving the 3D structure of the d-GCGAAGC mini-hairpin by NMR ( 40 ). The sequence was found to be folded back on itself between A 4 and A 5 and that all the sugars were in C2′-endo conformation. This compact molecule is stabilized by regular extensive base stacking interactions within each B-form helical strand of G 1 C 2 G 3 A 4 and A 5 G 6 C 7 and by two G-C base pairs and one G 3 -A 5 base pair (two hydrogen bonds) leaving the single base A 4 in the loop region. These highly stable hairpins also show high resistance to nucleases ( 40 ). Nuclease-dependent degradation has suggested that HPG11 is more resistant towards S1 nuclease than HPA11, reflecting the stability of -G- hairpin over -A- hairpin ( 25 ).
|
16855288_p25
|
16855288
|
UV-thermal denaturation experiments
| 4.487867 |
biomedical
|
Study
|
[
0.9993237257003784,
0.0003743796842172742,
0.000301924766972661
] |
[
0.9980742931365967,
0.00034034164855256677,
0.0014431842137128115,
0.00014207290951162577
] |
en
| 0.999996 |
The generic base sequences of the oligomers used in our study predict that since they are not fully self-complementary there exists a non-self-complementary residue (A or G) in the middle of the sequence intervening the complementary parts of the sequence. Of course the complementary ends of sequence can still interact and may form duplexes either intramolecularly (monomeric hairpin) or intermolecularly (bimolecular bulge duplex). The basis of formation of the bulge duplex structure is the quasipalindromic nature of the oligonucleotide. Since both sides of the central base (A or G) are self-complementary, the resulting intramolecular forms (hairpins) can have a single nucleotide loop and 5 bp stem (one AT and four GC base pairs).
|
16855288_p26
|
16855288
|
UV-thermal denaturation experiments
| 4.258769 |
biomedical
|
Study
|
[
0.9994614720344543,
0.00022766634356230497,
0.00031082204077392817
] |
[
0.9988545179367065,
0.000736563466489315,
0.00034106147359125316,
0.0000679183067404665
] |
en
| 0.999997 |
Further, a report from Fermandjian's group ( 41 ) on the formation of a DNA hairpin with a single residue loop (closed by a Watson–Crick G-C base pair) supports our prediction of existence of a single residue loop in mini-hairpins. NMR studies on d-AGCTTATC-ATC-GATAAGCT (-ATC-) encompassing the strongest topoisomerase II cleavage site in pBR322 DNA showed that the oligomer exists in hairpin form with a sheared A-C base pair to close the single base T loop. Further structural study using NMR, native PAGE, UV-melting, CD and restrained molecular dynamics on the -GAC- analog of the same oligomer reveal that -GAC- adopts a hairpin structure folded through a single residue loop. The residue A in the loop is closed by Watson–Crick type hydrogen bonds between G and C, however the base pair is not found planar but rather adopts a wedge-shaped geometry with the two bases stacked on top of each other in the minor groove. We predicted a similar situation in our studies on hairpin formation by HPA11 and HPG11 quasipalindromic oligonucleotides and their extended versions. The oligonucleotide either folds at the 5′- and 3′-sides of the central intervening residue or may be closed by a distorted Watson–Crick G.C base pair, which is further stabilized by extensive base stacking interactions along the 5′- and 3′-sides of the strands. For certain DNA hairpin loops a C.G closing base pair provides enhanced stability ( 42 ). Varani ( 43 ) has reviewed the structural, functional and thermodynamic aspects of exceptionally stable DNA and RNA hairpins. One of the classes of DNA and RNA hairpins containing teraloops of the GNRA family [sequence G- any nucleotide (N)- purine (R)-A] are highly stable due to the contacts of G and R not to each other but to the phosphates across the loop, and extensive base stacking interactions along the stem. Thus the actual loop is only a single residue (N). The loops are also stabilized by non-Watson–Crick base pairs and base-sugar contacts.
|
16855288_p27
|
16855288
|
UV-thermal denaturation experiments
| 4.643697 |
biomedical
|
Study
|
[
0.9989463686943054,
0.0005923922290094197,
0.00046126689994707704
] |
[
0.9841055274009705,
0.0007476194878108799,
0.01481815055012703,
0.0003287977597210556
] |
en
| 0.999997 |
Since, at this stage, we could not look into the structural details of the hairpin or bulge duplex species of the oligonucleotides used in this study, possibility of more than one residue in the hairpin loop cannot be ruled out.
|
16855288_p28
|
16855288
|
UV-thermal denaturation experiments
| 2.509654 |
biomedical
|
Study
|
[
0.9920823574066162,
0.0005515598459169269,
0.007366063538938761
] |
[
0.955615758895874,
0.04271416366100311,
0.0012045343173667789,
0.00046550322440452874
] |
en
| 0.999997 |
It is crucial here to refer to our earlier observation that none of the oligonucleotides under study showed the formation of G/C-quadruplexes like multistranded structures ( 25 ). This could be due to the fact that both the complementary -GGGG- and -CCCC- stretches separated by a base, are positioned on the same strand, leading them a scope of intramolecular or intermolecular base paired structures.
|
16855288_p29
|
16855288
|
UV-thermal denaturation experiments
| 4.048173 |
biomedical
|
Study
|
[
0.9993221759796143,
0.00015721093222964555,
0.0005206028581596911
] |
[
0.9979947805404663,
0.0016171553870663047,
0.00031804831814952195,
0.00007012915739323944
] |
en
| 0.999998 |
Further, CD spectroscopy, known to be extremely sensitive to small changes in mutual orientation of neighboring bases in an ordered or disordered duplex DNA, was used for the secondary structure analysis of the duplexes formed by the quasipalindromic complementary strands and their extended versions. CD spectra of the duplexes HP(C+G)11 and HP(C+G)21, prepared by mixing equimolar concentration (5 μM each) of respective strands in 20 mM sodium cacodylate buffer (pH 7.4) containing 100 mM NaCl and 0.1 mM EDTA are displayed in Figure 4a . Both the spectra are characterized by two prominent overlapping positive bands, which are usually not observed simultaneously for any canonical forms of DNA ( 44 ). It is interesting to note that the CD spectrum displayed here by the duplex HP(C+G)11 was found identical to the CD spectrum originated from HPG11 strand reported in our previous study ( 25 ). It is characterized by a strong negative band at 240 nm, crossover from negative to positive ellipticity at 247 nm, followed by two strong overlapping positive peaks near 265 and 285 nm. A careful look at the spectra in Figure 4a reveals that the structure formed by HP(C+G)11 show characteristics of both A- and B-type conformations. The spectrum displayed by the duplex HP(C+G)21 containing flanking sequences differs from HP(C+G)11 only at the shifted negative to positive cross over at 251 nm and positive CD peak with a 5 nm shift at 270 nm. Remarkably the second positive band at longer wavelength, belonging to both the duplex sequences occupies the same position at 285 nm. The amplitude of the positive peaks at 265 and 270 nm belonging to HP(C+G)11 and HP(C+G)21 duplex, respectively is almost equal, while it differs substantially at the longer wavelength positive band at 285 nm. The magnitude of the CD change at 285 nm positive band is associated with the sequence context of HP(C+G)11 and HP(C+G)21 duplexes and can be explained on the basis of few recent elegant reports, on the contribution of sequence to the CD profiles of A- and B-form of DNA ( 45 – 49 ).
|
16855288_p30
|
16855288
|
Circular dichroism experiments
| 4.358887 |
biomedical
|
Study
|
[
0.9993141889572144,
0.0003861458972096443,
0.00029974893550388515
] |
[
0.9989482760429382,
0.00023334719298873097,
0.0007194635691121221,
0.00009885084728011861
] |
en
| 0.999998 |
Coincidently, one of the studied sequences by Lindquist et al . ( 49 ) is a 10mer self-complementary oligonucleotide (TGGGGCCCCA) differing from our sequence only by 1 nt at the central position [i.e. TGGGG (A/G) CCCCA]. Notably this one base difference is not reflected in the CD spectra of the reported duplex and the duplex under study. Another independent study on similar lines ( 48 ) using the self-complementary d-GGGGCCCC sequence has concluded that the unusual spectra with simultaneous presence of A- and B-DNA type positive bands, contains features of A-like stacking of G-bases, and B-like stacking pattern for cytosines.
|
16855288_p31
|
16855288
|
Circular dichroism experiments
| 4.169794 |
biomedical
|
Study
|
[
0.999476969242096,
0.00016353702812921256,
0.00035946094430983067
] |
[
0.999077558517456,
0.00027520718867890537,
0.0005944345030002296,
0.00005277144009596668
] |
en
| 0.999996 |
Since we know that the sequences with guanine tracts forming parallel stranded tetraplexes are characterized by a positive CD signal at ∼260 nm ( 50 ), CD signal at 260–265 nm can be attributed to guanine–guanine stacking ( 46 ). Duplex sequences [HP(C+G)11, HP(C+G)21] studied by us contain a tract of four guanines, which gave positive CD signal at 265 nm. Following similar explanations given for sequences containing G-tract and C-tracts ( 45 , 51 ), it can be argued that the prefect duplexes formed by the quasipalindromic sequences under study have both A-type and B-type base pair segments. The effect of flanking sequences on the CD spectrum of -GGGGCCCC- tract has been reported ( 49 ). When the sequence is flanked by 5′-T/A or 5′-A/T, their CD showed a shoulder around 288 nm and a positive peak around 261–262 nm. With 5′-CAT flanking, it shows a weak positive peak at 288 nm, while the intensity of 266–268 nm positive band is somewhat enhanced. Significantly, the CAT sequence which is framed by 5′-CAT but where central G-tract was replaced by -ATGCAT- showed the spectrum typical of B-form DNA with a negative peak at 250 nm followed by a positive peak at 270 nm.
|
16855288_p32
|
16855288
|
Circular dichroism experiments
| 4.321396 |
biomedical
|
Study
|
[
0.9993498921394348,
0.00024355553614441305,
0.000406580074923113
] |
[
0.9992024302482605,
0.00022060881019569933,
0.0005125291645526886,
0.00006447736086556688
] |
en
| 0.999996 |
Our 5′- and 3′-flanks being -GCTCT and -GTACA (in HPA21/HPG21) and -TGTAC and -AGAGC (in HPT21/HPC21), when incorporated in 11mer quasipalindrome core -TGGGG(A/T/G/C)CCCCA- resulting into 21mer sequence (HPA21, HPT21, HPC21 and HPG21) showed a substantial effect on CD spectra of non-flanked sequences (11mers). The 5 nm red shift (265→270 nm) at the positive band of HP(C+G)21 is the consequence of incorporation of flanking sequence but it is still shown to retain A-like CD features ( 44 ). Watson–Crick base pairing between the complementary portions of HPC21 and HPG21 flanks may generate a small duplex segment. B-DNA stem contributes to the usual maximum at 285 nm ( 47 ). This maximum is more pronounced in case of HP(C+G)21 than HP(C+G)11, which concludes that the enhanced magnitude of 285 nm CD band is a result of increase in the ‘B’ character in HP(C+G)21.
|
16855288_p33
|
16855288
|
Circular dichroism experiments
| 4.361699 |
biomedical
|
Study
|
[
0.9993457198143005,
0.00031132003641687334,
0.0003430201322771609
] |
[
0.9992935657501221,
0.0002913288481067866,
0.0003374723019078374,
0.0000776041197241284
] |
en
| 0.999998 |
The present study is a follow up of our previous report ( 25 ) where the secondary structure of G and A strands of the duplex HP(C+G)11/HP(A+T)11 were investigated and were shown to exist in hairpin-duplex equilibria. The most interesting observation was a clear correlation of this hairpin-duplex equilibrium with A → B transition of DNA. The hairpin and duplex conformations exhibited an oligomer concentration dependence generating two different CD profiles of which the hairpin form was interpreted as the A-form, while the bulge duplex form was featured as B-form. Thinking on similar lines, we got interested to see whether the extended versions of duplex strands HPG21 and HPA21 also show oligomer concentration dependence on CD spectra as shown by their non-flanked version HPG11 and HPA11. For this purpose, CD spectra were recorded for the HPG21 sequence at varied strand concentrations (10–40 μM) . Such CD profiles depicted a strong oligomer concentration dependence, likewise the prominent 265 nm positive peak at 10 μM, disappeared with successive increase of strand concentrations upto 40 μM. The CD shoulder at 285 nm survived throughout the concentration range. Interestingly the spectra pass through an isoelliptic point at around 258 nm indicating more than one species in equilibrium. It is important to mention here that HPA21 also showed a similar strand concentration dependence on CD (data not shown). This concludes that like the non-flanked sequences (HPG11/HPA11) their extended versions (HPG21/HPA21) also undergo intramolecular (hairpin) → intermolecular (duplex) structural transition fairly reflected in A → B transition.
|
16855288_p34
|
16855288
|
Circular dichroism experiments
| 4.325859 |
biomedical
|
Study
|
[
0.9992928504943848,
0.00034370747744105756,
0.00036350011941976845
] |
[
0.9993771910667419,
0.00021444800950121135,
0.0003372051869519055,
0.00007125503179850057
] |
en
| 0.999998 |
The identical behavior shown by both the flanked and non-flanked duplex strands reveal a common structural feature present in HP(C+G)11/HP(C+G)21 sequences. At this stage it is important to mention the extensive work of other groups carried out on similar lines. Since our core sequence [TGGGG (A/T/C/G) CCCCA] of all duplexes share the same 5′- and 3′-terminal pentanucleotide stretches, with the sequence d-TGGGGCCCCA ( 49 ) and the -GGGG-/-CCCC- segments with d-GGGGCCCC ( 48 ) and d-CCCCGGGG ( 45 , 46 ), conformational similarities are anticipated. In all these reports the sequences have been the perfect palindromes (GGGGCCCC or CCCCGGGG). They were shown to form perfect duplexes with no signatures of hairpin or multistranded structures. Moreover the sequence d-TGGGGCCCCA did not show any oligomer concentration dependence on CD spectra ( 49 ). Thus it can be said that seemingly the quasipalindromic nature of our sequences created a space to exist in hairpin-duplex equilibrium. A-form DNA is less flexible than B-DNA and this rigidity is proposed to be of biological significance, since replication can occur at higher fidelity due to the stiffness of A-form structure ( 52 ).
|
16855288_p35
|
16855288
|
Circular dichroism experiments
| 4.299446 |
biomedical
|
Study
|
[
0.9994218349456787,
0.00023853141465224326,
0.00033964557223953307
] |
[
0.9990764856338501,
0.00025113471201620996,
0.0006045179325155914,
0.00006783020944567397
] |
en
| 0.999996 |
A study from Dickerson group ( 53 ) revealed unusual conformation for the dodecamer duplex formed by CATGGGCCCATG. It lies on a structural continuum along the transition between A- and B-DNA. The structure is an intermediate state of A → B helix transition. All sequences in nucleic acids database containing three or more GpG base steps have been crystallized in A-form ( 53 ). The crystal structure of CCCCGGGG possesses similar structural feature to that of GGGGCCCC ( 54 ). Thus as expected on the basis of above discussion, our quasipalindromes TGGGG(A/G)CCCCT containing three GpG steps should also adopt geometries closer to A- than B-DNA.
|
16855288_p36
|
16855288
|
Circular dichroism experiments
| 4.27856 |
biomedical
|
Study
|
[
0.9993714690208435,
0.00017918228695634753,
0.00044936922495253384
] |
[
0.999006450176239,
0.00039045518497005105,
0.0005469581228680909,
0.00005613068788079545
] |
en
| 0.999996 |
The X-ray study on induction of A → B transition in hexamer duplex d(CCCGGG) by nogalamycin appeared little interesting to us as, the DNA sequence used contains some elements common to our studied sequence ( 55 ).
|
16855288_p37
|
16855288
|
Circular dichroism experiments
| 3.060292 |
biomedical
|
Study
|
[
0.9955974221229553,
0.0003307705919723958,
0.0040718549862504005
] |
[
0.986677885055542,
0.012678996659815311,
0.0004087419656571001,
0.0002344042295590043
] |
en
| 0.999996 |
Based on the above mention experimental results, it can be said that since the individual strands of HP(C+G)11 and HP(A+T)11 can adopt intramolecularly folded and intermolecular linear duplex structures, their equimolar mixtures do not only hybridize to produce perfect duplexes but also species of hairpin, bulge duplex conformers.
|
16855288_p38
|
16855288
|
Circular dichroism experiments
| 3.94119 |
biomedical
|
Study
|
[
0.9983647465705872,
0.00018737019854597747,
0.0014479502569884062
] |
[
0.9885469675064087,
0.010797299444675446,
0.0005239421734586358,
0.00013179499364923686
] |
en
| 0.999996 |
In case of HP(C+G)21/HP(A+T)21 there rests a fair possibility that both the predicted forms (hairpin and duplex) are present simultaneously, thus formation of an extended bulge duplex or compact cruciform structure cannot be ruled out. Such a possibility is displayed in Figure 5b . It is clear that presence of flanking regions on 21mer sequences after hybridization with their complementary counterparts will generate 5 bp duplex regions on 5′- and 3′-termini of the strands, while the 11 nt core sequences will form the two hairpins (5 bp stem and single base loop) positioned opposite to each other. Such a simultaneous formation of two hairpins and paired flanking duplex regions shape into a cruciform structure. The possibility of such a four junction structure in HP(C+G)11/HP(A+T)11 is abridged due to lack of flanking nucleotides.
|
16855288_p39
|
16855288
|
Circular dichroism experiments
| 4.294513 |
biomedical
|
Study
|
[
0.9990788698196411,
0.0002842749818228185,
0.000636858690995723
] |
[
0.9986423850059509,
0.0010511328000575304,
0.00022142076340969652,
0.00008509333565598354
] |
en
| 0.999997 |
DNA sequences are now recognized to be structural determinants modulating the biological activity of genes. Perhaps the most important aspect of DNA structural variations is likely to be found in the mechanics of molecular recognition and manipulation by proteins. Our study presented here, imply that duplex formation at an imperfect inverted repeat sequence of HS4 of the β-globin LCR is restricted by the self-structuration of the relevant complements. As a result, hybridization of 11-base long quasipalindromic sequences generated not only the perfect (hetero-) duplexes but also the imperfect bulge (homo-) duplexes and hairpins via inter- and intramolecular Watson–Crick base pairing, while hybridization products of their extended versions 21mer flanking sequences were found to be in equilibrium with the perfect duplexes/bulge duplexes/cruciforms or hairpin species. The hybridized duplex segments also demonstrated the presence of interconvertible A- and B-DNA structural elements. Correlation of the intramolecular (hairpin) structure with the A-form and intermolecular (duplex) structure with B-form of DNA was concluded by CD signatures. Taken together these observations point out towards an important structural polymorphism, which could be biologically relevant. The conformational switching occurred in an oligomer concentration dependent manner might be considered as a structural motif to act as one of the regulatory elements in LCR. The human β-globin gene LCR, a dominant regulator of globin gene expression, is a contiguous piece of DNA with five tissue-specific DNase I HSs. Since the regions of HSs have a high density of transcription factor binding sites, structural interdependencies between HSs and different promoters may directly or indirectly regulate LCR functions. The DNA sequences of a SNP site studied here may contribute to form stable hairpin or cruciform structures at HS4 region of LCR suggesting that such structural polymorphism may cause variations in the DNase hypersensitivity.
|
16855288_p40
|
16855288
|
BIOLOGICAL IMPLICATIONS
| 4.6305 |
biomedical
|
Study
|
[
0.9990014433860779,
0.0007037229952402413,
0.00029484342667274177
] |
[
0.9984026551246643,
0.0006002281443215907,
0.0007022760692052543,
0.0002947940374724567
] |
en
| 0.999996 |
Literature is rich in studies showing that DNA is a dynamic molecule whose structure depends on the underlying nucleotide sequence and is influenced by the environment and the overall DNA topology. Important are the dynamic alterations in the structure of double helix including the generation and removal of non B-structures. Such structural polymorphism may be thought to regulate transcriptional activity in eukaryotic nucleus ( 56 , 57 ).
|
16855288_p41
|
16855288
|
BIOLOGICAL IMPLICATIONS
| 3.991656 |
biomedical
|
Study
|
[
0.9994677901268005,
0.00013367824431043118,
0.0003985851944889873
] |
[
0.9052383303642273,
0.02703000418841839,
0.06736952066421509,
0.000362170219887048
] |
en
| 0.999996 |
B-form is the major conformation of physiological DNA, while the A-form is the major conformation of RNA and can exist also for DNA under special conditions. The switch between A- and B-forms with a change in the structural geometries (folded unimolecular → linear biomolecular) is expected to have a spectacular role in the ability of the DNA segment to interact with proteins or other ligands. Implication of such structural transition has recently been identified ( 58 ). A minor groove-binding tract (MGBT) structural element of HIV-1 transcriptase is important for both replication frameshift fidelity and processivity. Interestingly the MGBT interactions occur in the DNA minor groove, where the DNA undergoes a structural transition from A-form to B-form DNA. Crystal structure of HIV-1 reverse transcripts complexed with double-stranded DNA also revealed that the template-primer has A-form and B-form regions separated by a significant bend 45° ( 59 ). The binding site of the nuclear single-stranded binding factor (NssBF) located in the LTR of the Drosophila 1731 retrotransposon mainly adopts two hairpin structures differing loop size, in slow equilibrium at pH 6.0. Two transcription factors bind only to the coding strand within the whole retrotransposon, suggesting that its structural flexibility could be associated with transcription ( 60 ). Transition from the B- to A-form of DNA is essential for biological functions as shown by the existence of A-form in many protein–DNA complexes ( 61 ).
|
16855288_p42
|
16855288
|
BIOLOGICAL IMPLICATIONS
| 4.648061 |
biomedical
|
Study
|
[
0.9992062449455261,
0.00046203259262256324,
0.0003317186201456934
] |
[
0.990996241569519,
0.0006305818096734583,
0.008117401041090488,
0.00025575130712240934
] |
en
| 0.999997 |
Our proposed model for the cruciform structure formed by HP(C+G)21 duplex is composed of two hairpins and a four arm junction . This single structure as discussed above exhibits segments of A- and B-DNA, such a possibility seem to be a mere consequence of nucleotide sequence ( 48 , 49 , 62 , 63 ). The formation of cruciforms is mostly favored in DNA sequences with inverted repeat symmetry, producing a discontinuity in regular DNA structure and therefore increasing the free energy of DNA molecules. However, cruciform extrusion relaxes superhelical strain, lowering the free energy of negative supercoiling ( 2 ). The frequency of occurrence of strong cruciform forming sequences has been reported in yeast and humans ( 64 ). Most recently Potaman and colleagues have reported ( 65 ) specific binding of Poly(ADP-ribose) Polymerase-1 (PARP-1) to cruciform hairpins. Interestingly, this nuclear protein differs from other cruciform-binding proteins by binding to hairpin tips rather than to four way junctions. The same study also indicated that PARP-1 can interact with the gene regulatory sequences by binding to promoter-localized cruciforms.
|
16855288_p43
|
16855288
|
BIOLOGICAL IMPLICATIONS
| 4.527684 |
biomedical
|
Study
|
[
0.9993451237678528,
0.0003365341981407255,
0.0003183577791787684
] |
[
0.9979360103607178,
0.00040717158117331564,
0.0015294475015252829,
0.00012749634333886206
] |
en
| 0.999997 |
Observation of cruciform DNA harbouring palindromic sequences and homologous duplex interaction to form four way junctions strengthens further the Gierer's hypothesis proposing that DNA may form branching structures analogous to t-RNA ( 66 ). Our comprehension of the role of hairpins, bulge duplexes and cruciform structural features formed at the SNP site in HS4 of the β-globin gene LCR has yet to be revealed. The discoveries described above give rise to a wealth of implications for future investigations relevant to sequence-specific structural heterogeneity within Watson–Crick base paired double helical molecules.
|
16855288_p44
|
16855288
|
BIOLOGICAL IMPLICATIONS
| 4.189749 |
biomedical
|
Study
|
[
0.9995844960212708,
0.00017013818433042616,
0.00024538012803532183
] |
[
0.985308825969696,
0.00139544066041708,
0.01311791967600584,
0.0001778350560925901
] |
en
| 0.999995 |
This paper in general concludes that the genomic quasipalindromic sequences containing G- and C-tracts may produce a discontinuity in regular DNA structure. These regions may fold back on themselves to form hairpins or cruciform structures (comprising of two hairpins). To sum up, the results of gel, UV-melting and CD analysis suggest that due to the propensity of oligonucleotides under study to acquire stable secondary structures other than duplex forms, their hybridization properties are restricted up to some extent. This accounts for the HP(C+G)11 and HP(A+T)11 sequences (expected to be perfect duplexes) to exist in equilibrium with hairpin, bulge duplex and perfect duplex forms. The hairpin forms display A- type CD spectra, while bulge duplexes are found to adopt B-form DNA. The CD spectra displayed contained both type of CD signatures. Presence of flanking sequences gives a possibility to HP(C+G)21/HP(A+T)21 to exist as a mixture of cruciform / bulge duplex and hairpin forms. Since, hairpins and bulge duplex forms represent A- and B-DNA form, respectively; cruciforms (comprising of two hairpins and duplex regions) render simultaneous presence of A- and B-type structural elements. To the best of our knowledge this is the first report where the possibility of a cruciform structure encompassing the elements of A- as well as B-DNA has been revealed, at a biologically relevant genomic site.
|
16855288_p45
|
16855288
|
CONCLUSIONS
| 4.457936 |
biomedical
|
Study
|
[
0.9993162155151367,
0.00038696362753398716,
0.000296824611723423
] |
[
0.9985979199409485,
0.000383784034056589,
0.0009008490014821291,
0.00011754446313716471
] |
en
| 0.999997 |
The usefulness of the work done in the paper, in a broad sense is information based. Structural polymorphism (hairpins, bulge duplexes, cruciforms etc.) and geometrical switching of DNA (A-form → B-form) exhibited by quasipalindromic regions within LCR of β-globin gene, highlight a careful understanding of the sequence dependent variations of the DNA structure. The knowledge of sequence-specific structural heterogeneity within Watson–Crick base paired double helical molecules might shed light on the mechanisms involved in transcriptional controlling of gene expression.
|
16855288_p46
|
16855288
|
PERSPECTIVES
| 4.103398 |
biomedical
|
Study
|
[
0.9996408224105835,
0.00010539698268985376,
0.0002538445987738669
] |
[
0.9894806146621704,
0.007509677205234766,
0.002812201390042901,
0.00019755169341806322
] |
en
| 0.999998 |
Cruciform DNA structures harbouring imperfect G- and C-tract palindromic sequences with simultaneous presence of A-and B-type DNA segments, could be used as a target for structure specific peptides / ligands or may serve as novel targets for pharmaceutical research. A protein could initially recognize a particular sequence from the shape of the DNA it binds to.
|
16855288_p47
|
16855288
|
PERSPECTIVES
| 3.870408 |
biomedical
|
Other
|
[
0.9983717799186707,
0.0005001288373023272,
0.0011281670304015279
] |
[
0.180419921875,
0.8088573813438416,
0.00999705120921135,
0.0007257215329445899
] |
en
| 0.999998 |
It is important to mention here that though both A- and B-forms of DNA have been largely studied by X-ray and NMR methods, the reasons for the preference for one conformation over the other are still unclear. The microscopic mechanism for the A → B conversion seem more difficult to understand in a situation like ours, where the A-form is only detected below 50 μM DNA concentration. Structural elucidation for the A-form, detectable only at micro-molar concentrations seems to be a difficult task and a challenging one indeed, for the structural biologists. We believe that such findings emphasize the importance of careful understanding of the molecular switching and a better analysis of the sequence dependent variations of the DNA structure. Our studies should endeavour to uncover structural details of nucleic acid structural transitions occurring at physiological conditions.
|
16855288_p48
|
16855288
|
PERSPECTIVES
| 4.173388 |
biomedical
|
Study
|
[
0.9996346235275269,
0.00014983068103902042,
0.00021552585531026125
] |
[
0.9905306696891785,
0.002302279695868492,
0.007029287982732058,
0.0001376771688228473
] |
en
| 0.999995 |
RNA chains can fold into complex secondary and tertiary structures, which often correspond to the minimum energy or equilibrium structure. Some RNAs, however, fold into long-lasting non-equilibrium conformations, which are known as metastable structures ( 1 – 8 ). Most of these structures are not biologically active and are thus termed misfolded ( 9 , 10 ). However, in a number of biological systems metastable structures exist that are actually not misfolded, but functionally important. In addition, a single RNA sequence can exhibit two catalytic activities resulting from two different structures ( 11 ).
|
16855293_p0
|
16855293
|
INTRODUCTION
| 4.166377 |
biomedical
|
Study
|
[
0.9992857575416565,
0.0002305052912561223,
0.0004838132008444518
] |
[
0.8224853873252869,
0.005592313129454851,
0.17157283425331116,
0.0003495337441563606
] |
en
| 0.999997 |
To understand how a folding RNA chain chooses between different alternative structures it is important to know which structural, thermodynamic and kinetic parameters control the folding of the various structural elements. Today, thermodynamic parameters of most of the RNA secondary structural elements are known ( 12 , 13 ), whereas kinetic parameters of RNA folding are scarce ( 8 , 14 – 17 ). It has been shown that the rate-determining step of hairpin formation is dependent on cancellation of the positive loop energy by the stacking interaction between the first closing base pairs ( 16 , 18 ) and that local hairpin formation is favoured over long-distance structural elements, because of the spatial proximity of the opposing base pairing partners ( 1 , 15 ). Little is known, however, about the effects of the nucleotide sequence and the size of hairpin loops and of the nature of the closing base pairs on folding kinetics. Even less is known about the effects of bulges, internal loops and other secondary structural elements.
|
16855293_p1
|
16855293
|
INTRODUCTION
| 4.384027 |
biomedical
|
Study
|
[
0.9991752505302429,
0.0004700313147623092,
0.0003546610532794148
] |
[
0.9669719338417053,
0.0005874978378415108,
0.03220539540052414,
0.0002351432922296226
] |
en
| 0.999995 |
Despite this lack of quantitative knowledge, great progress has been made in predicting folding routes of RNA using computer simulations, based on existing thermodynamic parameters and statistical polymer physics ( 2 , 4 , 19 – 25 ). These predictions, however, have rarely been verified experimentally. As a result it is still difficult to estimate which of the potential hairpins in a given RNA sequence will fold predominantly and which are kinetically disfavoured. Therefore, the prediction of a correct metastable structure in a given RNA molecule, even if it is suspected to have kinetically favourable metastable hairpins, has not always been straightforward ( 4 , 6 , 26 ) (J. H. A. Nagel, J. Møller-Jensen, C. Flamm, K. J. Öistämö, J. Besnard, I. L. Hofacker, A. P. Gultyaev, M. H. de Smit, P. K. Schuster, K. Gerdes and C. W. A. Pleij, manuscript submitted).
|
16855293_p2
|
16855293
|
INTRODUCTION
| 4.130479 |
biomedical
|
Study
|
[
0.9992024302482605,
0.0002567976771388203,
0.0005408244323916733
] |
[
0.7565466165542603,
0.006495084147900343,
0.2364530712366104,
0.000505200820043683
] |
en
| 0.999995 |
To determine kinetic parameters experimentally, we have developed an approach in which the kinetic folding ratios of two mutually exclusive hairpins in a given RNA sequence can be measured by structure probing. Although, this approach does not allow direct measurement of the hairpin-folding rate, it enables one to determine the effects of nucleotide substitutions, deletions and insertions. In turn, this allows us to assess, which sequence elements in an RNA chain are involved in the initiation of hairpin formation and to what degree. With these results it is possible to test current theoretical assumptions and to improve the predictive power of the RNA folding simulation programs.
|
16855293_p3
|
16855293
|
INTRODUCTION
| 4.180396 |
biomedical
|
Study
|
[
0.9995452761650085,
0.00023743057681713253,
0.00021718275093007833
] |
[
0.9986194372177124,
0.0006446255720220506,
0.0006616197642870247,
0.0000742551201255992
] |
en
| 0.999996 |
The RNA fragments were synthesized by IBA NAPS GmbH. The 5′ end labelling was done in 1× One Phor All plus buffer with 0.95 U of T4 Polynucleotide Kinase (Pharmacia) and incubated for 45 min at 37°C.
|
16855293_p4
|
16855293
|
5′ End 32 P labelling of RNA
| 3.835845 |
biomedical
|
Study
|
[
0.9989233613014221,
0.00041970895836129785,
0.0006568700191564858
] |
[
0.87260502576828,
0.12552295625209808,
0.0012027418706566095,
0.0006692769820801914
] |
en
| 0.999997 |
The heating and rapid cooling procedure for the RNAs was done in 50 mM Na cacodylate buffer (pH 7.2). First the sample was heated at 95°C for 2 min and then immediately placed into liquid nitrogen. Subsequently, the sample was slowly melted and used in the probing experiments ( 27 ).
|
16855293_p5
|
16855293
|
Kinetic trapping of RNA fragments
| 3.959384 |
biomedical
|
Study
|
[
0.9993430972099304,
0.00026360410265624523,
0.00039329970604740083
] |
[
0.9604222178459167,
0.03838149458169937,
0.0008830740116536617,
0.0003132819547317922
] |
en
| 0.999996 |
The acid denaturation of the RNA was done in 50 mM HCl at 0°C. Renaturation was achieved by adding 50 mM NaOH, 900 mM NaCl and 50 mM Na cacodylate buffer (pH 7.2). The pH was checked after each mixing procedure ( 27 ).
|
16855293_p6
|
16855293
|
Kinetic trapping of RNA fragments
| 3.926707 |
biomedical
|
Study
|
[
0.9992945194244385,
0.00023690467060077935,
0.00046855511027388275
] |
[
0.965808629989624,
0.033046212047338486,
0.0008846248383633792,
0.00026061665266752243
] |
en
| 0.999997 |
Thermodynamic equilibrium samples were obtained from kinetically trapped RNA fragments after an incubation of 30 min at 65°C, followed by slow cooling to 37°C and a final incubation of 2 h at 0°C, prior to structure probing.
|
16855293_p7
|
16855293
|
Kinetic trapping of RNA fragments
| 4.071029 |
biomedical
|
Study
|
[
0.9995133876800537,
0.0002201994211645797,
0.00026645249454304576
] |
[
0.9983603358268738,
0.0012116514844819903,
0.0003501654136925936,
0.00007790110976202413
] |
en
| 0.999995 |
Structure probing with RNases T1, T2 and V1 under native conditions was performed in 100 mM NaCl, 10 mM MgCl 2 and 50 mM Na cacodylate buffer (pH 7.2) in the presence of 10 µg tRNA per reaction mixture. The enzyme concentrations used were 1.25 U of RNase T1 (Promega), 0.5 U of RNase T2 (Promega) and 0.001 U of RNase V1 (Estonian Academy of Sciences) in 50 µl with incubation times of 5 and 15 min for the kinetically trapped and thermodynamic equilibrium samples. For the refolding experiments of the kinetically trapped RNAs, the RNA samples were incubated at 0°C for 0, 30, 60 and 120 min followed by a 10 min enzymatic probing incubation at 0°C. The thermodynamic equilibrium sample (∞) was used as a reference sample. Digestion with RNase T1 under denaturing conditions was done in 6 µl of 0.4% (w/v) Na citrate·2H 2 O, 0.14% (w/v) citric acid, 8 M urea and 0.4% (w/v) EDTA and 10 µg tRNA was added to 3 µl of sample RNA ( 32 P 5′ end labelled). The mixture was pre-incubated for 15 min at 55°C. Then 1.5 µl of RNase T1 (1 U) was added to the mixture, which was incubated for a further 20 min at 55°C. The alkaline ladder was made from the tested RNA sequence in a freshly prepared 25 mM Na 2 CO 3 /NaHCO 3 (1:9) buffer and heated for 2 min at 95°C. All probing mixtures were loaded on a 20% polyacrylamide sequencing gel containing 8 M urea, and detection was by autoradiography and phosphor imaging. The results from four to eight independent experiments were taken to calculate the folding ratios and standard deviation of the two mutually exclusive hairpins.
|
16855293_p8
|
16855293
|
Structure probing
| 4.253293 |
biomedical
|
Study
|
[
0.9993026256561279,
0.00042735482566058636,
0.00027000525733456016
] |
[
0.9992233514785767,
0.00028794663376174867,
0.00040612753946334124,
0.0000825819224701263
] |
en
| 0.999996 |
Candidate RNA sequences were designed manually to fold into either of two mutually exclusive hairpins. These two hairpins differ in their so-called ‘nucleation points’ or hairpin starting points by having different closing base pairs or loop sequences ( Table 1 ). In adition, differences beyond the five loop-proximal base pairs were introduced to prevent misfolding, duplex formation and to control the hairpin stability as required. The folding ratio is assumed to depend exclusively on the top of the hairpins, since the subsequent stacking interactions form approximately two orders of magnitude faster than the first closing interaction ( 16 , 28 , 29 ). Furthermore, the more distal base pairs are unlikely to be nucleation points of hairpin formation due to their unfavourable localization along the chain ( 14 , 25 , 29 ).
|
16855293_p9
|
16855293
|
Design of the experiment
| 4.207623 |
biomedical
|
Study
|
[
0.9994100332260132,
0.00027965064509771764,
0.0003103478520642966
] |
[
0.9992838501930237,
0.00026550967595539987,
0.0003928951337002218,
0.00005773705925093964
] |
en
| 0.999996 |
The computer program ‘Barriers’ ( 2 , 19 , 28 , 30 ) was then used to analyse the folding landscape of these candidate sequences . This enabled us to select those sequences having no additional significant stable hairpins and/or local energy minima in their folding landscape beyond the two desired ones. When necessary, further manual changes were introduced until the sequences met all design criteria.
|
16855293_p10
|
16855293
|
Design of the experiment
| 3.916031 |
biomedical
|
Study
|
[
0.9990683197975159,
0.00024096688139252365,
0.0006908124778419733
] |
[
0.9988763928413391,
0.0007333393441513181,
0.00032633758382871747,
0.0000638773781247437
] |
en
| 0.999998 |
The selected RNA sequences were synthesized and used in experiments where folding of the two competing hairpins from the single-stranded RNA was detected by secondary structure probing. To trap the RNA in the kinetically favoured conformation, it was denatured either by heat or by acid, followed by rapid cooling or a pH-jump to neutral pH, respectively (see Materials and Methods). Both methods gave identical results, indicating that the trapping procedure does not influence the kinetic competition between the two mutually exclusive hairpins, identical to earlier findings from our group ( 27 ).
|
16855293_p11
|
16855293
|
Design of the experiment
| 4.094952 |
biomedical
|
Study
|
[
0.9995076656341553,
0.0002550142235122621,
0.00023732225236017257
] |
[
0.9994040727615356,
0.00020874268375337124,
0.0003312534827273339,
0.00005597135896096006
] |
en
| 0.999997 |
This kinetically based competitional folding places strict experimental constraints on the design of the RNA sequences. First of all an individual RNA molecule, once folded into one of the two mutually exclusive hairpins should be prevented from refolding during the trapping and detection procedure into the competing, potentially more stable second hairpin . Otherwise one would detect the thermodynamically most stable hairpin and not necessarily the kinetically favoured one. The absence of this so-called ‘thermodynamic scrambling’ of the hairpins was tested for each RNA fragment with one or both trapping methods by following the refolding for 2–3 h at 0°C. Samples were taken at different time points and probed at 0°C. In none of the designed RNAs, the refolding into the thermodynamically more stable hairpin was complete after the incubation period. This indicated that during the probing time of 5, 10 and 15 min at 0°C, no significant thermodynamic scrambling occurred.
|
16855293_p12
|
16855293
|
Design of the experiment
| 4.175811 |
biomedical
|
Study
|
[
0.9993788003921509,
0.0002895490324590355,
0.0003316974616609514
] |
[
0.9993318915367126,
0.000257961597526446,
0.00035378537722863257,
0.000056291220971615985
] |
en
| 0.999997 |
Probing times of 5, 10 and 15 min were used to ensure that the enzyme concentration used would result in only a single cut in each RNA chain probed. This increase in probing time should result into a linear increase in the intensities of the probing bands observed, but should not lead to a significant decrease of the unprobed RNA fraction. For all RNA fragments this was indeed observed.
|
16855293_p13
|
16855293
|
Design of the experiment
| 4.008314 |
biomedical
|
Study
|
[
0.9993520379066467,
0.0002608948852866888,
0.00038704206235706806
] |
[
0.9987401366233826,
0.0009052854729816318,
0.0002720296324696392,
0.00008251050167018548
] |
en
| 0.999997 |
A further experimental constraint is that the two mutually exclusive hairpins should have distinct non-overlapping probing patterns. This means that the fragment lengths created by the RNases T 1 and T 2 for both the hairpin loops and single-stranded regions, should be unique and localized in a specific region in the separation gel to allow accurate determination of the folding ratios . This ensures that the measured intensity of a particular band can be directly correlated with its corresponding hairpin. In addition, quantitative differences in the accessibility to the RNases T 1 and T 2 of the individual unpaired nucleotides should be compensated for. This was achieved by comparing the probing efficiencies of the two mutually exclusive hairpins individually, using smaller RNA fragments harbouring only one of the two hairpins . RNase V 1 , probing paired nucleotides, was used as a control for the stem regions of the two hairpins.
|
16855293_p14
|
16855293
|
Design of the experiment
| 4.169281 |
biomedical
|
Study
|
[
0.9993971586227417,
0.0002799629874061793,
0.0003228534187655896
] |
[
0.9993816614151001,
0.00028198250220157206,
0.00028620162629522383,
0.0000501965478179045
] |
en
| 0.999997 |
Finally, to test and to minimize duplex formation, the concentration of the RNA was varied and/or a 10 to 100-fold excess of tRNA was added prior to both kinetic trapping procedures. No significant changes were observed (data not shown).
|
16855293_p15
|
16855293
|
Design of the experiment
| 3.219381 |
biomedical
|
Study
|
[
0.9978132247924805,
0.0004896395839750767,
0.0016971529694274068
] |
[
0.9974489808082581,
0.0018737409263849258,
0.0005278150201775134,
0.00014939320681151003
] |
en
| 0.999997 |
To test the design of the RNA fragments, first identical loops were introduced in the two mutually exclusive hairpins JN1C and JN2C, with the loop sequences GC AAAA GC and GC AGAA GC , respectively ( Table 1 ). The folding kinetics of the two hairpins are expected to be the same, because the loops and the closing base pairs constitute identical nucleation points. The experimental kinetic ratio for the JN1C fragment suggested a slight preference for the 5′ end hairpin ( Table 1 ). However, this is still within experimental error, because the overall reproducibility of the trapping and probing procedure is ∼5–10%.
|
16855293_p16
|
16855293
|
Experimental determination of folding ratios
| 4.12371 |
biomedical
|
Study
|
[
0.9992988109588623,
0.0003011003427673131,
0.0004000146291218698
] |
[
0.9995406866073608,
0.0002091598289553076,
0.00019379204604774714,
0.00005647565922117792
] |
en
| 0.999998 |
In the thermodynamic equilibrium experiment the two hairpins were also found in similar amounts, so thermodynamic scrambling during the trapping procedure could not be excluded. With the JN2C fragment the differences in Δ G between the two hairpins was enlarged while maintaining identical loops and closing base pairs. Furthermore, the presence of a G residue in the loop enabled us to include RNase T 1 (specific for unpaired G residues) as a structure probe. The kinetic experiment again yielded equal amounts of the two hairpins, while a clear thermodynamic shift towards the more stable 3′-hairpin was observed in the thermodynamic folding experiment, as expected . These findings strongly support the validity of the experimental approach.
|
16855293_p17
|
16855293
|
Experimental determination of folding ratios
| 4.187566 |
biomedical
|
Study
|
[
0.9994482398033142,
0.0002721667115110904,
0.00027958935243077576
] |
[
0.9993979930877686,
0.0002716997405514121,
0.00026021531084552407,
0.00007013024878688157
] |
en
| 0.999998 |
In RNA so-called stable tetra-loops exist, in which an additional gain in free energy of hairpin formation is obtained by specific stacking and hydrogen bond interactions in the loop ( 12 ). The two main classes are the GNRA and YNMG tetra-loops (N can be any nucleotide, R is either A or G, M is either C or A and Y is either C or U) ( 31 ). To test the effect of a stable tetra-loop upon hairpin formation, a GC AGAA GC loop was compared against a stable GC GGAA GC (GNRA) tetra-loop (JN2D, Table 1 ). We expected that the hairpin with the GGAA stable tetra-loop would out-compete the AGAA loop, because it stabilized the first stacking interaction by 3 kcal/mol ( 13 ). Surprisingly, however, the hairpins folded in a equal ratio ( Table 1 ). The experimental result indicates that the possibility of forming a GGAA tetra-loop does not accelerate folding of the corresponding hairpin.
|
16855293_p18
|
16855293
|
Effects of stable tetra-loops
| 4.191719 |
biomedical
|
Study
|
[
0.9993323683738708,
0.00026936002541333437,
0.00039825536077842116
] |
[
0.9994869232177734,
0.00022403558250516653,
0.00023228504869621247,
0.00005673465057043359
] |
en
| 0.999998 |
Previous computer simulations indicated that the folding ratio of a particular structure correlates directly with its number of nucleation points ( 2 , 3 ). To examine the effects of multiple nucleation points and to test an additional GNRA stable tetra-loop, four more RNA sequences were designed (JN1LH, JN2LH, JN3LH and JN4LH) ( Table 1 ). These four sequences can either fold into a two-hairpin structure without stable tetra-loops or into a single hairpin (rod-like), with (JN3LH and JN4LH) or without (JN1LH and JN2LH) the GCGA stable tetra-loop .
|
16855293_p19
|
16855293
|
Effects of stable tetra-loops
| 4.089279 |
biomedical
|
Study
|
[
0.9993758797645569,
0.00018868158804252744,
0.00043541842023842037
] |
[
0.9995318651199341,
0.00024691951693966985,
0.00017493095947429538,
0.00004637304664356634
] |
en
| 0.999996 |
The kinetic folding experiment of the JN1LH and JN2LH fragments showed a 1:2 folding ratio between the rod-like structure, with one nucleation point and the two-hairpin structure with two nucleation points, as predicted . The fact that the folding ratio was identical for both fragments confirmed that these directly reflect folding kinetics. This despite the fact that the thermodynamically most stable conformation, at 37°C, is the rod-like structure in JN1LH, and the two-hairpin one in JN2LH.
|
16855293_p20
|
16855293
|
Effects of stable tetra-loops
| 4.195077 |
biomedical
|
Study
|
[
0.9994282126426697,
0.0002966637839563191,
0.0002750544808804989
] |
[
0.9993438124656677,
0.00032051693415269256,
0.0002616798738017678,
0.0000739032548153773
] |
en
| 0.999997 |
Remarkably, the same kinetic folding ratios were experimentally obtained for the JN3LH and JN4LH sequences, in which a GCGA stable tetra-loop was introduced into the rod-like structure. This confirms the results obtained with the JN2D sequence ( Table 1 ), showing that the extra thermodynamic stability of the stable tetra-loops does not influence their folding kinetics.
|
16855293_p21
|
16855293
|
Effects of stable tetra-loops
| 4.103473 |
biomedical
|
Study
|
[
0.9991518259048462,
0.0002549565106164664,
0.0005932075437158346
] |
[
0.9995112419128418,
0.0002521126007195562,
0.00018434842058923095,
0.00005227405927143991
] |
en
| 0.999997 |
Next, we addressed the possible role of several primary and secondary structure elements like closing base pairs, loop sizes, loop sequences and internal loops, on hairpin-folding kinetics. With JN3A and JN3B ( GU GAAA GC versus GC GAAA GC) the influence of the close stacking interaction on the kinetic folding was examined. We expected that a U–G closing base pair would form less efficiently than a C–G closing pair, due to its smaller Δ G contribution upon stacking. Similarly, we expected that the change in loop size from four to five bases in JN4A ( GC AAAAA GC versus GC AAAA GC) and in JN4B ( GC AAGAA GC versus GC AGAA GC) would result in slower folding rates. The kinetic probing experiment, however, showed equal ratios for both the closing base pair and loop size RNA sets ( Table 1 ), indicating that the folding kinetics were unaffected.
|
16855293_p22
|
16855293
|
Effects of stem and loop sequences
| 4.163598 |
biomedical
|
Study
|
[
0.9993801116943359,
0.00030351901659742,
0.00031634781043976545
] |
[
0.9994658827781677,
0.00018076322157867253,
0.0002906549780163914,
0.00006272691825870425
] |
en
| 0.999996 |
The importance of the nucleotide composition of a loop for the rate of hairpin formation has been shown in DNA hairpins ( 32 – 34 ). In DNA folding, pyrimidine-rich loops (T-loops) fold faster than A-rich-loops, because in the latter case single-stranded A stacks have to be disrupted prior to folding. A similar effect is expected for RNA hairpin formation. Therefore, the JN6A sequence was designed, containing a 5′ end hairpin loop with only pyrimidines and a mutually exclusive 3′ end purine-rich hairpin loop ( Table 1 ). The pyrimidine-rich 5′ end hairpin motif was derived from the kinetically favourable 5′ end metastable hairpin I of the hok mRNA (J. H. A. Nagel, J. Møller-Jensen, C. Flamm, K. J. Öistämö, J. Besnard, I. L. Hofacker, A. P. Gultyaev, M. H. de Smit, P. K. Schuster, K. Gerdes and C. W. A. Pleij, manuscript submitted) ( 35 , 36 ). The experimental results show that this pyrimidine-rich loop is indeed the faster folder, as it partially out-competed the purine-rich loop of the 3′-hairpin ( Table 1 ).
|
16855293_p23
|
16855293
|
Effects of stem and loop sequences
| 4.263652 |
biomedical
|
Study
|
[
0.999537467956543,
0.00023255324049387127,
0.00022993251332081854
] |
[
0.9991687536239624,
0.0002583805762697011,
0.0004950393340550363,
0.00007783366163494065
] |
en
| 0.999997 |
The potential kinetic effect of an internal loop on hairpin formation rates was tested with the JN5A and JN5B sequences. In these RNAs the 5′ end hairpin contains a GG mismatch after the first two closing base pairs, while the mutually exclusive 3′ end hairpin forms an uninterrupted stem. The experimentally determined ratio is approximately equal in both the JN5A and JN5B RNA fragments. This was unexpected, because the interrupted base pair zippering in the 5′ hairpin was presumed to slow down its folding. Apparently, zippering through and beyond the internal loop is still faster than disruption of the initial closing interactions.
|
16855293_p24
|
16855293
|
Effects of stem and loop sequences
| 4.217413 |
biomedical
|
Study
|
[
0.9993256330490112,
0.00031201340607367456,
0.00036234164144843817
] |
[
0.9993317723274231,
0.00031603971729055047,
0.0002804176474455744,
0.00007167659350670874
] |
en
| 0.999996 |
To directly compare theoretical predictions and experiments, we extracted predicted folding ratios from folding simulations using the recently developed program ‘Kinfold’ from the Vienna RNA package ( ). This comparison is important, because thus far, little direct experimental verification existed to justify the currently used kinetic parameters. The Kinfold program performs stochastic simulations of folding and refolding behaviour of RNA sequences [see Refs ( 2 , 28 ) for details]. The program models the process of RNA folding as a Markov Chain at single base pair resolution via a Monte Carlo process, meaning that the smallest change or move set in the simulation is an addition or disruption of a single base pair. To calculate the folding rates between two neighbouring states the Metropolis rule was used. Each simulation for an RNA sequence was started in the open chain conformation (i.e. with no base pairs present) and ran until one of the two stable states was reached. At least 2000 such trajectories were computed for each designed molecule to get an accurate estimate of the folding ratio.
|
16855293_p25
|
16855293
|
Folding simulations
| 4.163599 |
biomedical
|
Study
|
[
0.9994712471961975,
0.0003091571561526507,
0.00021962345635984093
] |
[
0.9990835189819336,
0.00017422412929590791,
0.0006679948419332504,
0.00007420706242555752
] |
en
| 0.999994 |
The program ‘Barriers’ ( 21 , 28 ) was used to construct the folding landscape and barrier trees for the Kinfold program, from an energy sorted list of all possible suboptimal conformations generated by the program RNAsubopt ( 30 ) . To aid the calculations, the list of suboptimal conformations was limited to a predefined energy range so that those suboptimal conformations, which contributed <1% to either the folding landscape or the folding ratios were eliminated from the calculation. This is to prevent endless calculations for non-contributing conformations.
|
16855293_p26
|
16855293
|
Folding simulations
| 4.115423 |
biomedical
|
Study
|
[
0.9994353652000427,
0.00019932424766011536,
0.00036534015089273453
] |
[
0.9985886216163635,
0.0009766565635800362,
0.0003714833874255419,
0.00006315184145933017
] |
en
| 0.999997 |
The program Barriers identifies all local minima and the energy barriers between them according to a single base pair move set. Thus, the calculated folding funnels are ideally separated by a single ‘saddle point’ (the lowest energy barrier between the two funnels). In our RNA fragments the folding ratios of the two mutually exclusive hairpins can then be calculated, provided that there are only two major nucleation points in the RNA chain, each leading to one of the two possible hairpins . This turned out to be a difficult condition to fulfil, because the experimentally required high stability of the hairpins demanded relatively long sequences, thereby increasing the risk of creating multiple nucleation points.
|
16855293_p27
|
16855293
|
Folding simulations
| 4.205194 |
biomedical
|
Study
|
[
0.9994198083877563,
0.00022079615155234933,
0.00035944744013249874
] |
[
0.9988744854927063,
0.0008141868747770786,
0.0002449744497425854,
0.00006635471800109372
] |
en
| 0.999998 |
The simulations with the ‘Kinfold’ program for our RNA sequences illustrate the difficulties encountered with this type of computer predictions. While the computations and experiments are in excellent agreement for JN2C and JN5B sequences they differ significantly for JN1C, JN4A and JN4B RNAs. This could be due to an additional hairpin observed in the calculations but not in the experiments, which creates an additional nucleation point favouring the 3′ hairpin. Theoretically this could result in the 2:1 ratio in favour of the 3′-hairpin, which was predicted for the JN1C, JN4A and JN4B fragments ( Table 1 ).
|
16855293_p28
|
16855293
|
Folding simulations
| 4.136464 |
biomedical
|
Study
|
[
0.9994571805000305,
0.0002214486594311893,
0.000321294239256531
] |
[
0.9994292855262756,
0.00022003857884556055,
0.00029450058354996145,
0.00005609290019492619
] |
en
| 0.999994 |
The folding simulations initially included the additional energy for the stable tetra-loop as a favourable kinetic parameter. However, on the basis of the experimental results for the JN2D, JN3LH and JN4LH RNAs, containing GNRA tetra-loops, a subsequent simulated folding analysis was done excluding this energy as a folding parameter. This gave folding ratios, which were similar to the experimental results for the JN3LH and JN4LH sequences and improved the predicted ratios for the JN2LH, JN3B and JN5B ( Table 1 ). However, for the JN2D and JN5A RNAs, correct prediction of the folding ratio seemed to require the tetra-loop parameter at 0°C ( Table 1 ). For the JN2D, this could be due to the prediction of an additional hairpin favouring the 3′-hairpin when the stable tetra-loop energy was excluded.
|
16855293_p29
|
16855293
|
Folding simulations
| 4.156803 |
biomedical
|
Study
|
[
0.999356210231781,
0.00028118633781559765,
0.00036266646930016577
] |
[
0.9994737505912781,
0.00015998036542441696,
0.0003065476194024086,
0.000059740003052866086
] |
en
| 0.999997 |
Though, the simulation program does not take the nucleotide content of the loop into account, the predicted JN6A ratios favoured the purine-rich 3′ end loop at 0°C, in contrast with the experimental results. In addition, when the tetra-loop energies were included, it predicted an alternative stable tetra-loop and a G bulge ( Table 1 ).
|
16855293_p30
|
16855293
|
Folding simulations
| 4.133063 |
biomedical
|
Study
|
[
0.9995080232620239,
0.00020014010078739375,
0.00029182713478803635
] |
[
0.9995254278182983,
0.0002301941131008789,
0.00018578326853457838,
0.000058686709962785244
] |
en
| 0.999996 |
In this paper a set of RNA sequences were designed that enabled us to experimentally determine relative folding rates of two mutually exclusive RNA hairpins, by kinetic trapping and structure probing procedures. The advantage of this combination of techniques is that it is applicable to a large variety of RNA hairpins without the need to modify the RNA with, for instance, fluorescent labels. In addition, it can be used with a variety of buffers and reaction conditions. Although enzymatic structure probing was chosen here, one can in principle use chemical modifications or other detection techniques as well. The methods chosen allowed us to address a number of questions in relation to sequence specific folding kinetics of RNA hairpins, both experimentally and computationally.
|
16855293_p31
|
16855293
|
DISCUSSION
| 4.095214 |
biomedical
|
Study
|
[
0.9995144605636597,
0.00025203480618074536,
0.00023354360018856823
] |
[
0.9990843534469604,
0.0002401721285423264,
0.0006143641076050699,
0.00006114214920671657
] |
en
| 0.999997 |
The results obtained with the JN1C and 2C sequences show that after kinetic trapping of the RNA, the ratio of the two mutually exclusive hairpins can be accurately determined and that no thermodynamic scrambling or refolding takes place during the detection time of the probing experiment.
|
16855293_p32
|
16855293
|
DISCUSSION
| 4.025142 |
biomedical
|
Study
|
[
0.9992375373840332,
0.00028284103609621525,
0.00047974076005630195
] |
[
0.9989222288131714,
0.0006866816547699273,
0.00031373166712000966,
0.00007739571447018534
] |
en
| 0.999996 |
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