Molecular dynamics structures of peptide nucleic acidÆDNA hybrid in the wild-type and mutated alleles of Ki-ras proto-oncogene Stereochemical rationale for the low affinity of PNA in the presence of an A C mismatch Thenmalarchelvi Rathinavelan and Narayanarao Yathindra Department of Crystallography and Biophysics, University of Madras, Guindy Campus, Chennai, India Institute of Bioinformatics and Applied Biotechnology, ITPB, Bangalore, India Peptide nucleic acids (PNAs) stand out from the rest of the nucleic acid mimetic, in that they consist of an uncharged N-(2-aminoethyl) glycine (Fig. 1) backbone scaffold [1,2]. These enable them to defy protease and nuclease digestion, and therefore serve as promising contenders as antigene and antisense agents [3–10]. PNA mediated transcription inhibition occurs either by strand invasion or by conventional triplex forma- tion [1,2]. In the former, PNA displaces one of the strands of the DNA duplex by forming Watson and Crick (WC) base pairs leading to a PNAÆDNA duplex (duplex invasion) or by forming WC and Hoogsteen Keywords enthalpy-entropy contribution; fluctuating A .C mismatch hydrogen bond; mismatch containing PNAÆDNA hybrid; point mutation Correspondence N. Yathindra, Institute of Bioinformatics and Applied Biotechnology, G-05, Tech Park Mall, ITPB, Bangalore-560 066, India Fax: +91 80 2841 2761 Tel: +91 80 2841 0029 E-mail: yathindra@ibab.ac.in (Received 13 April 2005, revised 3 June 2005, accepted 14 June 2005) doi:10.1111/j.1742-4658.2005.04817.x The low affinity of peptide nucleic acid (PNA) to hybridize with DNA in the presence of a mismatch endows PNA with a high degree of discriminat- ory capacity that has been exploited in therapeutics for the selective inhibi- tion of the expression of point-mutated genes. To obtain a structural basis for this intriguing property, molecular dynamics simulations are carried out on PNAÆDNA duplexes formed at the Ki-ras proto-oncogene, compri- sing the point-mutated (GAT), and the corresponding wild-type (GGT) codon 12. The designed PNA forms an A. C mismatch with the wild-type sequence and a perfect A T pair with the point mutated sequence. Results show that large movements in the pyrimidine base of the A C mismatch cause loss of stacking, especially with its penultimate base, concomitant with a variable mismatch hydrogen bond, including its occasional absence. These, in turn, bring about dynamic water interactions in the vicinity of the mismatch. Enthalpy loss and the disproportionate entropy gain associ- ated with these are implicated as the factors contributing to the increase in free energy and diminished stability of PNAÆDNA duplex with the A C mismatch. Absence of these in the isosequential DNA duplex, notwith- standing the A C mismatch, is attributed to the differences in topology of PNAÆDNA vis-a ` -vis DNA duplexes. It is speculated that similar effects might be responsible for the reduced stability observed in PNAÆDNA duplexes containing other base pair mismatches, and also in mismatch con- taining PNAÆRNA duplexes. Abbreviations DD wt , DNA duplex with A .C mismatch; DD mut , DNA duplex with A .T pair; LNA, locked nucleic acid; MD, molecular dynamics; PD wt , PNAÆDNA duplex with A. . .C mismatch; PD mut , PNAÆDNA duplex with Watson and Crick A .T pairing; PNA, peptide nucleic acid; RMSD, root mean square deviation; T m , melting temperature; WC, Watson and Crick. FEBS Journal 272 (2005) 4055–4070 ª 2005 FEBS 4055 base pairs leading to a PNAÆDNAÆPNA triplex (triplex invasion). Duplex strand invasion mechanism has the advantage of targeting any sequence in a DNA duplex, without the stringent prerequisite of a polypurine tract as in the conventional triplex-mediated transcription repression. PNAÆDNA duplexes are more stable than their iso- sequential DNA duplexes at moderate salt levels, as a consequence of reduced electrostatic repulsion caused by the conspicuous absence of phosphates in the PNA strand [11,12]. Another distinctive characteristic of PNAÆDNA complex formation has been the high degree of discrimination for sequence selectivity with the complementary strand of DNA [11,13] given the significantly less stable nature of the PNAÆDNA duplex in the presence of even a single mismatch. This is found to be true for a variety of mismatches [14–16], a property that is in sharp contrast to mismatch con- taining DNA duplexes. This unique feature is utilized to detect point mutation [17–24], selective amplifica- tion ⁄ suppression of DNA target using PCR clamping [25–27], and selective suppression of replication [28] and gene expression [29,29a] by suitable choice of base sequence in PNA. A case in point is its utility in selec- tive inhibition of gene expression in the mutational hotspots of ras proto-oncogenes. Normal ras proto- oncogenes express p21, an important signal transduc- tion protein, and a single mutation at one of the few critical positions of ras proto-oncogenes results in a single amino acid substitution in p21 [30] causing malignancy [31]. One such point mutation, occurring in codon 12 of the Ki-ras proto-oncogene, replaces GGT with GAT [32] (capped region in Scheme 1) in one of the alleles of pancreatic cells. This leads to pan- creatic cancer [32], as Asp (GAT) replaces Gly (GGT) in p21. A selective inhibition of the mutated Ki-ras proto-oncogene can be effected by designing a PNA so as to form a mismatch (PD wt ) with the wild-type allele (unmutated proto-oncogene), and a perfect WC base pair (PD mut ) with the mutated allele (mutated proto- oncogene). The logic is that the former, in view of the mismatch, is rendered a less stable PNAÆDNA complex promoting normal expression, while the latter (mutated) forms a stable PNAÆDNA duplex (without mismatch) causing inhibition of gene expression. Using this strategy, a differential proliferation effect of the wild-type (with A C mismatch), and mutated (with A T pair) alleles of Ki-ras proto-oncogene, has been reported recently [29,29a]. Needless to say, a structure- based rationale is obligatory to comprehend the causa- tive factors for the destabilization of PNA ÆDNA in the presence of mismatch compared to DNA duplex. Inci- dentally, no structural information either from NMR, X-ray crystal structure or modelling is available for PNAÆDNA duplex with a mismatch. It is in this con- text, molecular dynamics (MD) simulations have been carried out on PNAÆDNA and DNA duplexes, formed out of a sequence present in the Ki-ras proto-onco- gene, in the presence and absence of an A C mis- match. Results reveal that enthalpic loss and the concomitant, but disproportionate entropic gain due to interrupted stacking, fluctuating nature of the hydro- gen bond and water organization in the vicinity of the mismatch might be the contributing factors for the increase in free energy and diminished stability of PNAÆDNA vis-a ` -vis DNA duplex. Fig. 1. Schematic representation of a section of peptide nucleic acid (PNA) chain along with notations for the backbone and side chain torsion angles: a(C6¢-N1¢-C2¢-C3¢), b(N1¢-C2¢-C3¢-N4¢), c(C2¢-C3¢- N4¢-C5¢), d(C3¢-N4¢-C5¢-C6¢), e(N4¢-C5¢-C6¢-N1¢), n(C5¢-C6¢-N1¢-C2¢), v1(C8¢-C7¢-N4¢-C3¢), v2(N9 ⁄ N1-C8¢-C7¢-N4¢)andv3(C4 ⁄ C2-N9 ⁄ N1-C8¢-C7¢). Planar peptide unit is enclosed in a rectangle. Peptide hydrogen atom alone is shown for clarity. Effect of A .C mismatch in PNAÆDNA and DNA duplexes T. Rathinavelan and N. Yathindra 4056 FEBS Journal 272 (2005) 4055–4070 ª 2005 FEBS Results For convenience of discussion, and to be consistent with the strategy of designing of PNA for gene suppres- sion through PNAÆDNA duplex formation (see above), the 15mer PNAÆDNA duplexes formed with an A C mismatch (wild-type allele) and with WC A T pairing (mutated allele) are referred to as PD wt and PD mut , respectively (Scheme 1). Likewise, the corresponding isosequential DNA duplexes are referred to as DD wt (with an A C mismatch) and DD mut (with A T pair), respectively. Because base stacking and base pair- ing interactions are the major sources of stabilization of nucleic acid duplexes, their comparison, especially in the vicinity of the mismatch in PD wt compared with DD wt duplex may give clues towards deciphering the origin of the destabilization and hence, diminution of the melting temperature (T m ) in the former. Base stacking in the vicinity of A C mismatch in PNAÆDNA and DNA duplexes Intra strand base stacking at the AC(6–7) (Fig. 2A) & CC(7–8) steps (Fig. 2B) of the DNA strand, and GA(23–24) (Fig. 2C) and AT(24–25) (Fig. 2D) steps of the PNA strand, flanking the A24 C7 mismatch in PD wt (Scheme 1), and the corresponding AT(6–7) & TC(7–8) steps of the DNA strand (Fig. 2E,F), and GA(23–24) & AT(24–25) (Fig. 2G,H) steps of the PNA strand in PD mut (Scheme 1) are monitored. Base stacking at the CC(7–8) step of PD wt (Fig. 2B), and the TC(7–8) step of PD mut (Fig. 2F) of the DNA strand show significant differences. This is due to con- siderable movement of cytosine (C7) of the A24 C7 mismatch of PD wt , leading to large fluctuations in its interaction with the adjacent pyrimidine base (C8). This results in hardly any stacking between them. Only occasionally, C5-H group of cytosine (C8) over- laps with C7 and, O2 of C7 overlaps with C8. On the other hand, sustained stacking persists by way of par- tial overlap of T7 and C8 (Fig. 2F) at the correspond- ing TC(7–8) step of PD mut . A totally unstacked situation is seldom seen here indicating that occur- rence of an A24 C7 mismatch brings about signifi- cant reduction in adjacent base stacking in PD wt compared to PD mut . On the other hand, stacking at the AC(6–7) step in the DNA strand of PD wt is retained during the entire simulation in spite of the large movement of C7 (Fig. 2A). This occurs due to the coordinated move- ments of C7 and A6 which ensure stacking through- out. Similarly, stacking persists at the corresponding AT(6–7) step in PD mut (Fig. 2E) through interaction of A6 with either the six-member ring of T7 or through the methyl group and ⁄ or O4 of T7. Thus, base stack- ing prevails at the AC(6–7) step (Fig. 2A) of PD wt , and the AT(6–7) step of PD mut (Fig. 2E). Likewise, the extent of intra strand base stacking at the GA(23–24) and AT(24–25) steps of the PNA strand in both PD wt (Fig. 2C,D) and PD mut (Fig. 2G,H) is essentially sim- ilar. Thus, A24 C7 mismatch leads to an almost complete loss of stacking only at the CC(7–8) step (PD wt ), while the stacking is maintained in the other steps that flank the mismatch. Scheme 1. Sequences encompassing codon 12 (capped) of the Ki-ras proto-oncogene of wild type (wt) and mutated (mut) alleles. Bold-italic regions in both wild-type and mutated sequences represent the PNAÆDNA duplex. Mismatch (wild-type) and the corresponding ideal WC base pairs (mutated) are underlined. The C- and N-termini of the PNA are considered as equivalent to 3¢ and 5¢ ends of a nucleic acid chain, respectively. T. Rathinavelan and N. Yathindra Effect of A C mismatch in PNAÆDNA and DNA duplexes FEBS Journal 272 (2005) 4055–4070 ª 2005 FEBS 4057 It is clear from Fig. 3A–D that presence of an A C mismatch in DD wt , seemingly does not influence adja- cent base stacking at the mismatch site. Although stacking at the CC(7–8) step is found to be only margi- nal in DD wt during the first 220 ps, quite similar to that seen in the PD wt , it is enhanced significantly beyond 220 ps, so much so that almost a complete overlap of adjacent pyrimidines is observed (Fig. 3B). Stacking interactions at the neighbouring AC(6–7), GA(23–24) and AT(24–25) steps of the A24 C7 mis- match site are also maintained (Fig. 3A,C,D). It is noteworthy that although stacking at the AC(6–7) step fluctuates, a complete loss of stacking is seldom found (Fig. 4A). Stacking persists either through the overlap A B C D E F G H Fig. 3. Stereo diagram of adjacent bases at various steps flanking the A24 .C7 mis- match in DD wt : (A) AC(6–7); (B) CC(7–8); (C) GA(23–24) and (D) AT(24–25), and their equivalent steps in DD mut : (E) AT(6–7); (F) TC(7–8); (G) GA(23–24) and (H) AT(24–25). Notice that stacking prevails in all the steps, both in DD wt and DD mut . C7 and A24 involved in A .C mismatch in DD wt and the equivalent T7 and A24 in DD mut are col- oured red. Trajectories corresponding to every 20 ps are shown. E F G H A B C D Fig. 2. Stereo diagram of adjacent bases at various steps flanking the A24 .C7 mis- match in PD wt : (A) AC(6–7); (B) CC(7–8); (C) GA(23–24) and (D) AT(24–25), and their equivalent steps in PD mut : (E) AT(6–7); (F) TC(7–8); (G) GA(23–24) and (H) AT(24–25). Note the interruption of the stack at the CC(7–8) step (B) in PD wt , while base stack- ing prevails at all the steps in PD mut (E–H). Large movements of C7 at CC(7–8) step (B) are apparent. C7 and A24 bases involved in A .C mismatch in PD wt and, the equivalent T7 and A24 bases in PD mut are coloured red. Trajectories corresponding to every 20 ps are shown. Effect of A .C mismatch in PNAÆDNA and DNA duplexes T. Rathinavelan and N. Yathindra 4058 FEBS Journal 272 (2005) 4055–4070 ª 2005 FEBS of the amino group of C7 with A6 (Fig. 4B) or through the overlap of the six-member ring of C7 with A6 (Fig. 4C). Thus, unlike in PD wt (Fig. 2B), uninter- rupted stacking prevails at all the steps of DD wt . Interestingly, the extent of stacking at the TC(7–8) step of DD mut with A T pair (Fig. 3F), is similar to that at the CC(7–8) step of DD wt (Fig. 3B). Further- more, it is evident that although the exact mode of stacking interactions at the AT(6–7) (Fig. 3E), GA(23– 24) (Fig. 3G) and AT(24–25) (Fig. 3H) steps in DD mut appear to be different from the equivalent steps in DD wt (Fig. 3A,C,D), the degree or extent of stacking is comparable. This suggests that the stacking interaction persists in the adjacent steps of both DD wt and DD mut . On the other hand, as noted above, stacking is inter- rupted in PNAÆDNA duplex with an A C mismatch. Variation of A C mismatch hydrogen bond in PNAÆDNA and DNA duplexes Fluctuations in the position of C7 of the A24 C7 mismatch in PD wt discussed above are also found to influence the nature of A24 C7 mismatch hydrogen bond. It is found that hydrogen bond fluctuates between N6(A24) N3(C7) and ⁄ or N6(A24). O2(C7) (Fig. 5). As C7 approaches A24, it engages in N6(A24) N3(C7) hydrogen bonding, and when C7 moves away from A24 along the major groove, the other possible hydrogen bonding schemes emerge (Fig. 5A–C). Extreme movement of C7 away from A24 can even result in the absence of both the hydro- gen bonds (Fig. 5D). These are apparent in Fig. 5E. MD simulations extended up to 4 ns further substanti- ates the variable nature of the hydrogen bond (Fig. 5). These clearly indicate the absence of a stable hydrogen bond for the A24 C7 mismatch in PD wt . In sharp contrast, a stable N1(A24) N4(C7) hydro- gen bond (Fig. 6B) prevails in DD wt , although the ini- tial N6(A24) N3(C7) hydrogen bond (Fig. 6A) lasts for a short duration (200 ps) (Fig. 6C–F). The transi- tion to the favoured hydrogen bond occurs as a result of movement of A24 rather than C7 (of the DNA strand) as found in PD wt and persists till the end of 4 ns dynamics. Further, A C mismatch hydrogen bond in DD wt is different from that found in PD wt (Fig. 5). An earlier MD simulation (just over 100 ps) based on NMR data on DNA duplex, pointing to the Ki-ras proto-oncogene having an A23 C8 mismatch (Scheme 1) instead of A24 C7 as in the present study, has indicated the possibility of all the three schemes for A23 C8 mismatch hydrogen bonding [33], but without preference for any one of them. However, it is found here that A24 C7 favours N1(A24) N4(C7) hydrogen bond. In any case, the present analysis clearly points to the greater changeability and destabilization of the A C mismatch hydrogen bond in PNAÆDNA than in DNA duplex. As expected, these bring forth significant varia- bleness in the water interactions surrounding the mis- match. Water interaction in the vicinity of A C mismatch Figure 7A–L depicts the nature of water interaction in the neighbourhood of A24 C7 mismatch in PD wt . Water interaction along the minor groove side of A24 C7 mismatch is conserved to the extent that either N1(A24) or O2(C7) or both, are involved in interaction with water. This is true irrespective of Fig. 4. Stacking interactions seen at AC(6–7) step of DD wt . Note the prevalence of stacking interaction (B and C) almost throughout dynamics (see also text) despite the fluctuations. Complete loss of stacking interaction is seldom seen (A). T. Rathinavelan and N. Yathindra Effect of A C mismatch in PNAÆDNA and DNA duplexes FEBS Journal 272 (2005) 4055–4070 ª 2005 FEBS 4059 the presence or absence of N6(A24) N3(C7) and⁄ or N6(A24) O2(C7) hydrogen bond. On the other hand, water interaction along the major groove is influenced by the nature of the mismatch hydrogen bond. When N3(C7) is not involved in hydrogen bond with N6(A24), it engages itself in a variety of interactions with water along the major groove side as shown in Fig. 7A,E,F,I. In the absence of both N6(A24) N3(C7) and N6(A24) O2(C7) hydrogen bonds due to the displacement of the C7 towards the major groove, N3(C7) and O2(C7), both are engaged in interaction with water (Fig. 7D). These are demon- strative of significant fluctuations in the water structure in the vicinity of A24 C7 mismatch in PD wt . In con- trast, such variation is not observed in DD wt due to the strong preference for N1(A24) N4(C7) hydrogen bond (Fig. 7M–T). As a result, N6(A24) and N4(C7) are involved in a variety of water interaction on the major groove side (Fig. 7N–T). Similarly, N3(A24), N3(C7) and O2(C7) are also engaged in water interac- tion most of the time (Fig. 7N–T). Thus, it is apparent that water interaction associated with the atoms parti- cipating in A24 C7 mismatch does not show fluctu- ation as in the case of PD wt . Fig. 5. Interaction between the A24 (blue) and C 7 mismatch bases in PD wt (A–D) and variation of N6(A24). . .N3(C7) and N6(A24) .O2(C7) hydrogen bond distances (F & H), and angles (G & I) over 4 ns dyna- mics. Large movement of C7 and the asso- ciated variable hydrogen bonding pattern for A24. . .C7 mismatch are clear from the superposition (E). Effect of A .C mismatch in PNAÆDNA and DNA duplexes T. Rathinavelan and N. Yathindra 4060 FEBS Journal 272 (2005) 4055–4070 ª 2005 FEBS Conformation of the PNA strand in PNAÆDNA duplexes Like in DNA, backbone conformation of the PNA scaffold is governed by six backbone torsion angles, a(C6¢-N1¢-C2¢-C3¢), b(N1¢-C2¢-C3¢-N4¢), c(C2¢-C3¢- N4¢-C5¢), d(C3¢-N4¢-C5¢-C6 ¢), e(N4¢-C5¢-C6¢-N1¢) and f(C5¢-C6¢-N1¢-C2¢). These are found to be confined to the trans ⁄ gauche – , gauche + , gauche + , gauche + , near cis and trans range of conformations, respectively, in both PD mut and PD wt (Fig. S1A–F of Supplementary material). The side chain torsion angles, v1(C8¢-C7¢- N4¢-C3¢), v2(N9 ⁄ N1-C8¢-C7¢-N4¢) and v3(C4 ⁄ C2- N9 ⁄ N1-C8¢-C7¢) favour the cis, trans ⁄ gauche – and gauche + conformations, respectively (Fig. S1G–I of Supplementary material). It is noteworthy that both backbone, as well as side chain, conformations of the PNA strand observed in the present study generally fall in the same range of conformational angles seen in the crystal structures of PNAÆDNA duplex [34] and (PNA) 2 ÆDNA triplex [35]. These are also broadly similar to the results obtained from earlier MD simu- lations on PNAÆDNA complexes [36,37]. Some differ- ences seen from the NMR structure may be due to under-determination of the backbone structure by NMR as acknowledged by the authors [38]. Inciden- tally, a designed PNA analogue with b ¼ gauche + region, similar to that observed in the current investi- gation, readily forms complex with both DNA and RNA [39–41]. A B C D EF Fig. 6. Hydrogen bonding schemes (A and B) observed for the A24 .C7 mismatch in DD wt . Variation of hydrogen bond distances (C and E) and angles (D and F) over 4 ns dynamics. Note the strong preference for N1(A24) .N4(C7) hydrogen bond beyond 220 ps (C and D). T. Rathinavelan and N. Yathindra Effect of A C mismatch in PNAÆDNA and DNA duplexes FEBS Journal 272 (2005) 4055–4070 ª 2005 FEBS 4061 Base backbone hydrogen bonds and a(N1¢-C2¢) and e(C5¢-C6¢) correlation in the PNA strand Interestingly, a near-neighbour bond correlation between the torsion angles a(N1¢-C2¢)ande(C5¢-C6¢) associated with the peptide unit is recognized. It is observed that whenever a undergoes a conformation change from the most preferred trans ⁄ gauche – to the gauche + conforma- tion, a concomitant change occurs in e from a cis to a trans conformation (Fig. 8) as found earlier [42]. These ensure stacking as well as the WC hydrogen bond (Fig. 9). Other transitions lead to a totally unstacked situation (data not shown). Further, the (gauche + , trans) conformational state for (a,e) promotes an intramolecu- lar O6¢ H-N2 (G) hydrogen bond between guanine and the carbonyl of the peptide (Fig. 10I). However, this is not possible for the (trans ⁄ gauche – , cis) conforma- tion as O6¢ orients towards the solvent with the amide (N1¢) hydrogen pointing inside the helix. On the other hand, this facilitates in the formation of hydrogen bond with N3 of purines and O2 of pyrimidines either directly (Fig. 10A,C,E,G) or through water molecules Fig. 7. Interaction of water (orange) with A24. . .C7 mismatch in PD wt (A–L) and DD wt (M–T) during the dynamics. Variation in hydration pattern in PD wt (A–L) depending on the A24 .C7 mismatch hydrogen bond- ing is readily apparent. Effect of A .C mismatch in PNAÆDNA and DNA duplexes T. Rathinavelan and N. Yathindra 4062 FEBS Journal 272 (2005) 4055–4070 ª 2005 FEBS (Fig. 10B,D,F,H). Interestingly, water mediated N3 N1¢ [34] and O2 N1¢ [34,35] interactions are found in the minor groove side of the PNAÆDNA duplex [34] and (PNA) 2 ÆDNA tripl ex [35] crysta l structures. PNAÆDNA duplex structure Average structure of the central 11mer of PD wt over 2.5 ns dynamics is shown in Fig. 11A. Root mean square deviation (RMSD) of the entire trajectory (2.5 ns) with respect to the average structure lies in the range  0.7–2.5 A ˚ for both PD wt and PD mut . Average value of helical twist corresponding to the central 11mer is  27° in both PD wt and PD mut leading to a 13-fold duplex. This is similar to that observed in an NMR study of a PNAÆDNA hybrid [38]. Average value of rise, slide, X-displacement and propeller twist correspond to values around 3.3 A ˚ , )1.2 A ˚ , )3.7 A ˚ and )10.7°, respectively, for PD wt , and 3.3 A ˚ , )1.3 A ˚ , )4.0 A ˚ and )10.6°, respectively, for PD mut . Average widths of minor and major grooves are around 9.5 A ˚ and 25 A ˚ in both PD wt and PD mut . Sugar puckers in DNA strands favour the C2¢ endo conformation in both PD wt and PD mut . Interestingly, C7 involved in A C mismatch seems to favour the C4¢ exo sugar pucker, although C2¢ endo is seen dur- ing the dynamics (data not shown). In general, stacking interaction is nearly similar in both PD mut and PD wt (data not shown) except at steps on either side of the mismatched A C hydrogen bond. DNA duplex structure Average structure of the central 11mer of DD wt over 2 ns dynamics is shown in Fig. 11B. RMSD of the entire trajectory (2 ns) corresponding to DD wt and DD mut varies from 1.2–2.1 A ˚ and 1.0–2.8 A ˚ , respect- ively, with respect to their average structure. Even Fig. 8. Correlation between the backbone torsion angles, a(N1¢-C2¢) and e(C5¢-C6¢)inPD wt (red) and PD mut (black). Notice the prefer- ence for (a,e) . (trans ⁄ gauche – ,nearcis) compared to (a,e) . (gauche + , trans) conformation. Fig. 9. Stereo plots illustrating the stacking interaction at the GC step when (a,e) . (trans ⁄ gauche – , near cis) (A and B) and (a,e) . (gauche + , trans) (C and D) conformations. O6¢(G). . .N2(G) hydrogen bond (C and D) is shown in dotted line (see also Fig. 10 and text). Hydrogens at C2¢,C3¢,C5¢ and C8¢ are not shown for clarity. T. Rathinavelan and N. Yathindra Effect of A C mismatch in PNAÆDNA and DNA duplexes FEBS Journal 272 (2005) 4055–4070 ª 2005 FEBS 4063 though RMSD is rather large for DD mut during the first 500 ps of the dynamics, it stabilizes later. RMSD of DD mut and DD wt falls between 1.0 and 2.0 A ˚ , beyond 500 ps representing the equilibrium state. The overall conformation of the helix is of B type. Average value of the major groove width is 16.8 A ˚ for DD wt and 18.0 A ˚ for DD mut , while the average widths of the minor groove are 11.4 A ˚ and 11.3 A ˚ for DD wt and DD mut , respectively. Fig. 10. Dependence of backbone .base hydrogen bond interactions in PNA on a and e correlation. Note that hydrogen bond between N1¢ (backbone) and base (O2 ⁄ N3) may be direct (A,C,E,G) or through water (B,D,F,H) when (a,e) . (trans ⁄ gauche – , near cis) and N1¢. . .N1¢ repeat is compact (5.5 A ˚ ). Direct hydrogen bond between O6¢ and N2 (I) is seen for (a,e) . (gauche + , trans) when N1¢. . .N1¢ repeat is extended (6.5 A ˚ ). Hydro- gens at C2¢,C3¢ and C5¢ are not shown for clarity. Effect of A .C mismatch in PNAÆDNA and DNA duplexes T. Rathinavelan and N. Yathindra 4064 FEBS Journal 272 (2005) 4055–4070 ª 2005 FEBS [...]... nature of the structure and interactions of nucleic acids, and especially so, in discerning the comparative in uence ` of base pair matches vis-a-vis normal ones Choice of the aforementioned sequence is because of the proven efficacy of the designed PNA to down-regulate the gene expression of the mutated allelle, while sustaining the transcription of the wild-type allele [29,29a] The present investigation... advantage of in the selective inhibition of gene expression of a point -mutated gene by appropriate design of a PNA [29,29a] Factors that contribute to the sharp decrease in free energy could arise from (a) reduced binding enthalpy caused by the loss of hydrogen bond and weak stacking and also (b) entropic and enthalpic contribution resulting from the nature of interaction of water in the vicinity of mismatch... N3(C7) and N6(A24) O2(C7) in PDwt Further simulation (1 ns) indicates that even at 400 K, the fluctuating nature of the mismatch hydrogen bond (data not shown) prevails in PDwt In contrast, a stable N1(A24) N4(C7) hydro4066 T Rathinavelan and N Yathindra gen bond is favoured throughout the dynamics in DDwt leading to an uninterrupted stacking at the steps flanking the mismatch The difference in the A... duplex DNA contributes an element of instability resulting in lowering of the melting temperature and increase in free energy Surprisingly, the presence of a mismatch in PNAÆDNA duplexes [11,14–16] has a pronounced effect in reducing the Tm, FEBS Journal 272 (2005) 4055–4070 ª 2005 FEBS Effect of A .C mismatch in PNAÆDNA and DNA duplexes with a free energy penalty of about 15 kJÆmol)1 per base pair... [45,46] and tRNA [47,48] The above deductions are also expected to hold true for other mismatch containing PNAÆDNA duplexes, wherein a similar drastic reduction in Tm accompanied by increase in free energy is reported [16], although the nature of the stacking, mismatch hydrogen bond and its fluctuating character in these cases may be governed to some extent by the sequences flanking the mismatch Interestingly,... 3DNA: a software package for the analysis, rebuilding and visualization of threedimensional nucleic acid structures Nucleic Acids Res 31, 5108–5121 Supplementary material T Rathinavelan and N Yathindra Fig S1 Bar diagram illustrating the normalized frequency of different backbone (A–F) and side chain (G–I) torsion angles of the PNA strand of PDwt (red) and PDmut (black) Fig S2 Partial charges for the different... chain (Fig S3E–H of the Supplementary material) Periodic box of TIP3P waters and 14 Na+ counter ions to neutralize the charge on the DNA strands of the hybrids are added using LEaP module of amber 6 This results in 4294 and 4379 number of water molecules for PDmut and PDwt systems, respectively, and periodic boxes of sizes ˚ ˚ ˚ ˚ ˚ ˚  48 A ·  51 A ·  77 A and  50 A ·  51 A ·  77 A are obtained... investigation is the first report on the structure and dynamics of a PNAÆDNA duplex with a mismatch The results of the analysis are also compared with the MD simulations on isosequential DNA duplexes in the presence (DDwt) and absence (DDmut) of an A C mismatch Our results point to considerable fluctuations in cytosine base (C7) of the A C mismatch in PDwt leading to transition between the two possible... chem.msu.su/gran/gamess/index.html) and RESP [60] module of amber 6, because the same is not available in the amber standard library Electrostatic potential calculated using the HF ⁄ 6–31G* basis set (pc gamess) is used in the calculation of RESP charges (Figs S2 and S3A–D of the Supplementary material) It is noteworthy that the partial charges for the various atoms of bases are very similar to when they are part of DNA... hydrogen bond may therefore be attributed to the significant differences in the topological features, namely, minor and major groove widths and X-displacement ˚ ( 9.5,  25,  )4 vs  11,  18,  )1 A) of PNAÆDNA and DNA duplexes It is noteworthy that all of these A C mismatch hydrogen bonds (Figs 5 and 6) noticed during the dynamics of PNAÆDNA and DNA duplexes are found in the crystal structure of ribosomal . Molecular dynamics structures of peptide nucleic acidÆDNA hybrid in the wild-type and mutated alleles of Ki-ras proto-oncogene Stereochemical. hydrogen pointing inside the helix. On the other hand, this facilitates in the formation of hydrogen bond with N3 of purines and O2 of pyrimidines either directly