N. Yathindra

Short oligodeoxynucleotides with d(G-C)n sequence do not assume left-handed conformation in high salt conditions.

d(G-C)n oligodeoxynucleotides (with n varying from 3 to 7) were studied by the circular dichroism technique in 5 M-NaCl. Contrary to what was previously found with the d(C-G)n series in the same solvent, the left-handed double-stranded Z-conformation appears less stable than the B-form for low n values. The influence of base sequence on the relative stability of B- and Z-conformations for the two series is discussed.

Flexibility Of DNA In 2:1 Drug-DNA Complexes - Simultaneous Binding Of Two DAPI Molecules To DNA

Simultaneous binding of two DAPI molecules in the minor groove of (dA)15.(dT)15 B-DNA helix has been simulated by molecular mechanics calculations. The energy minimised structure shows some novel features in relation to binding of DAPI molecules as well as the flexibility of the grooves of DNA helices. The minor groove of the helix expands locally considerably (to 15 angstroms) to accommodate the two DAPI molecules and is achieved by positive propeller twisting of base pairs at the binding site concomitant with small variations in the local nucleotide stereochemistry. The expansion also brings forth simultaneously a contraction in the width of the major groove spread over to a few phosphates. These findings demonstrate another facet of the flexible stereochemistry of DNA helices in which the local features are significantly altered without being propagated beyond a few base pairs, and with the rest of the regions retaining the normal structure. Both the DAPI molecules are engaged in specific hydrogen bonds with the bases and non specific interactions with phosphates. Stacking interactions of DAPI molecules between themselves as well as with sugar-phosphate backbone contribute to the stability of the complex. The studies provide a stereochemical support to the experimental findings that under high drug-DNA ratio DAPI could bind in the 2:1 ratio.

An innate twist between Crick’s wobble and Watson-Crick base pairs

Non-Watson-Crick pairs like the G·U wobble are frequent in RNA duplexes. Their geometric dissimilarity (nonisostericity) with the Watson-Crick base pairs and among themselves imparts structural variations decisive for biological functions. Through a novel circular representation of base pairs, a simple and general metric scheme for quantification of base-pair nonisostericity, in terms of residual twist and radial difference that can also envisage its mechanistic effect, is proposed. The scheme is exemplified by G·U and U·G wobble pairs, and their predicable local effects on helical twist angle are validated by MD simulations. New insights into a possible rationale for contextual occurrence of G·U and other non-WC pairs, as well as the influence of a G·U pair on other non-Watson-Crick pair neighborhood and RNA-protein interactions are obtained from analysis of crystal structure data. A few instances of RNA-protein interactions along the major groove are documented in addition to the well-recognized interaction of the G·U pair along the minor groove. The nonisostericity-mediated influence of wobble pairs for facilitating helical packing through long-range interactions in ribosomal RNAs is also reviewed.

A study of the interaction of DAPI with DNA containing AT and non-AT sequences--molecular specificity of minor groove binding drugs.

The binding specificity of DAPI to DNA has been probed by analysing its interactions with DNA octamers consisting of different base sequences, which include adenine, guanine, 2-amino adenine and inosine, using molecular mechanics methods. Presence of AT and non-AT base pairs in the immediate vicinity of the binding site, containing AT and non-AT base pairs is also investigated. Results show that DAPI most prefers to bind to homopolymer of AT, and least to the duplex containing alternating GC bases. DAPI interacts with homopolymeric duplexes in two possible orientations related by 180 degrees with nearly same affinity. Affinity of DAPI towards DNA comprising the modified bases, inosine and 2-amino adenine, is in between these extremities. The binding affinity is reduced to some extent by the occurrence of non AT bases flanking the four base paired binding region. An interesting revelation is that one can visualise DAPI to form a hydrogen bond with O2 of cytosine indicating that the 2-amino group of purines does not per se sterically preclude DAPI from residing in the minor groove of B-DNA helix. On the other hand, repulsive nature of electrostatic interactions that prevail at the minor groove consequent to the presence of these sequences contribute decisively in preventing further diffusion of the drug. Thus, electrostatics, rather than hydrogen bonding to bases, seemingly play an important role in determining the specificity of interaction. The retention of drug binders in the minor groove and therefore recognition, is governed by the combined effect of these various forces.

Studies on the cross stand hydrogen bonds in DNA double helices

A systematic analysis has been carried out to examine all the stereochemically possible bifurcated hydrogen bonds including those of cross strand type between propeller twisted base pairs in DNA double helices by stereochemical considerations involving base pairs alone and by molecular mechanics studies on dimer and trimer duplexes. The results show that there are limited number of combinations of adjacent base pairs that would facilitate bifurcated cross-strand hydrogen bond (CSH). B-type helices concomitant with negative propeller twist seem to be more favored for the occurrence of CSH than canonical A-type helices because of slide in the latter. The results also demonstrate that helices with appropriate sequences may possess continuous run of these propeller twist driven cross strand hydrogen bonds indicating that they may in fact be considered as yet another general structural feature of DNA helices.

Molecular dynamics structures of peptide nucleic acid·DNA hybrid in the wild‐type and mutated alleles of Ki‐ras proto‐oncogene

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 discriminatory capacity that has been exploited in therapeutics for the selective inhibition of the expression of point‐mutated genes. To obtain a structural basis for this intriguing property, molecular dynamics simulations are carried out on PNA·DNA duplexes formed at the Ki‐ras proto‐oncogene, comprising 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 associated with these are implicated as the factors contributing to the increase in free energy and diminished stability of PNA·DNA duplex with the A…C mismatch. Absence of these in the isosequential DNA duplex, notwithstanding the A…C mismatch, is attributed to the differences in topology of PNA·DNA vis‐à‐vis DNA duplexes. It is speculated that similar effects might be responsible for the reduced stability observed in PNA·DNA duplexes containing other base pair mismatches, and also in mismatch containing PNA·RNA duplexes.

The Left-Handed Z-DNA Conformation in Oligodeoxynucleotides Containing Different Amounts of AT Base Pairs: A Far UV Circular Dichroism Study

A number of fully self-complementary oligodeoxynucleotides have been synthesized and examined for their ability to assume the left-handed Z-DNA conformation in high salt solutions. The B- and Z-forms are identified by circular dichroism spectra, covering both the long- (220-300 nm) and short-wavelength (185-220 nm) regions, the latter showing CD bands very useful for identifying the sense of the helix winding. The main results of the study can be summarized as follows: a) sequences composed by AT and CG blocks do support the B to Z transition, even when the AT contents amounts to 50%; b) the occurrence of consecutive purine-purine or pyrimidine-pyrimidine dyads does not inhibit the B to Z transition, although a stronger reduction of water activity is required; c) (AC)n and (GT)n containing oligonucleotides do undergo the B to Z transition in solution; d) a millimolar quantity of Ni2+ concomitant with 5 M NaClO4 is found to be very effective in bringing about the B to Z transition in most of the sequences considered in this study.

Stereochemical effects of methylphosphonate in B- and Z-DNA helices: variation in hydrophobicity and effective widths of grooves

Stereochemical effects of methylphosphonate (MP) in B-DNA and Z-DNA duplexes are studied through molecular mechanics approach. Duplexes of different lengths, tetramers, hexamers, dodecamers are examined to assess the interstrand and intrastrand electrostatic effects due to MPs vis-a-vis phosphates. A variety of models which include duplexes with alternating S-MP and R-MP, alternating phosphate and MP and, duplexes possessing MPs in only one of the strands, are examined by considering both the S- and R-stereoisomers. Majority of the calculations are performed with CG sequences to delineate factors responsible for the stability of B- and Z-DNA, as well as B-->Z-DNA transition under nonionic conditions. The results show that both B- and Z-DNA duplexes are energetically favoured in the presence of MP due to overwhelming reduction in intrastrand as well as interstrand electrostatic repulsive interactions. The effect is distinct in oligomers longer than tetramers. Comparison of energetics of MP B- and Z-DNA duplexes suggests that an oligodeoxynucleotide such as d(CG)6 with all phosphates replaced by MPs may favour equally both B- and Z-DNA conformations. The analysis further provides an estimate of electrostatic interactions, operating at the grooves under a variety of conditions. Several specific and localised effects due to S-MP and R-MP are seen at CG and GC steps in various B-DNA and Z-DNA models. S-MP in B-DNA reduces the effective major groove width by nearly 3 A hence denying access to the functional groups of endonucleases thereby enhancing the resistance of MP-DNA to enzymatic digestion. Further, methyl groups of MP render the surface of the DNA helix to be significantly hydrophobic which may explain higher permeability of MP-DNA in membranes as well as its less soluble nature in aqueous media.

Relative Stability of B and Z Structures in Oligodeoxynucleotides with Different Alternating Base Sequence and Length

The conformation of several oligodeoxynucleotides with alternating purine-pyrimidine sequences in aqueous salt solution has been studied by means of CD technique. Although the solution conditions should favor the Z-conformation, only C-G sequences are found in this structure. (G-C)n sequences with n up to 5 do not assume the left-handed conformation. The concatamer obtained with d(AT)3(CG)3 in high salt solution was found in B conformation whereas the concatamer obtained with d(TA)3(CG)3 in the same conditions showed alternating blocks of B and Z structures. The effect of base-stacking in this behavior is discussed.

Selective Preference of Parallel DNA Triplexes Is Due to the Disruption of Hoogsteen Hydrogen Bonds Caused by the Severe Nonisostericity between the G*GC and T*AT Triplets.

Implications of DNA, RNA and RNA.DNA hybrid triplexes in diverse biological functions, diseases and therapeutic applications call for a thorough understanding of their structure-function relationships. Despite exhaustive studies mechanistic rationale for the discriminatory preference of parallel DNA triplexes with G*GC & T*AT triplets still remains elusive. Here, we show that the highest nonisostericity between the G*GC & T*AT triplets imposes extensive stereochemical rearrangements contributing to context dependent triplex destabilisation through selective disruption of Hoogsteen scheme of hydrogen bonds. MD simulations of nineteen DNA triplexes with an assortment of sequence milieu reveal for the first time fresh insights into the nature and extent of destabilization from a single (non-overlapping), double (overlapping) and multiple pairs of nonisosteric base triplets (NIBTs). It is found that a solitary pair of NIBTs, feasible either at a G*GC/T*AT or T*AT/G*GC triplex junction, does not impinge significantly on triplex stability. But two overlapping pairs of NIBTs resulting from either a T*AT or a G*GC interruption disrupt Hoogsteen pair to a noncanonical mismatch destabilizing the triplex by ~10 to 14 kcal/mol, implying that their frequent incidence in multiples, especially, in short sequences could even hinder triplex formation. The results provide (i) an unambiguous and generalised mechanistic rationale for the discriminatory trait of parallel triplexes, including those studied experimentally (ii) clarity for the prevalence of antiparallel triplexes and (iii) comprehensive perspectives on the sequence dependent influence of nonisosteric base triplets useful in the rational design of TFO’s against potential triplex target sites.