Thermodynamics of DNA duplexes with adjacent G.A mismatches.

The sequence 5'-d(ATGAGCGAAT) forms a very stable self-complementary duplex with four G.A mismatch base pairs (underlined) out of ten total base pairs [Li et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 26-30]. The conformation is in the general B-family and is stabilized by base-pair hydrogen bonding of an unusual type, by favorable base dipole orientations, and by extensive purine-purine stacking at the mismatched sites. We have synthesized 13 decamers with systematic variations in the sequence above to determine how the flanking sequences, the number of G.A mismatches, and the mismatch sequence order (5'-GA-3' or 5'-AG-3') affect the duplex stability. Changing A.T to G.C base pairs in sequences flanking the mismatches stabilizes the duplexes, but only to the extent observed with B-form DNA. The sequence 5'-pyrimidine-GA-purine-3', however, is considerably more stable than 5'-purine-GA-pyrimidine-3'. The most stable sequences with two pairs of adjacent G.A mismatches have thermodynamic parameters for duplex formation that are comparable to those for fully Watson-Crick base-paired duplexes. Similar sequences with single G.A pairs are much less stable than sequences with adjacent G.A mismatches. Reversing the mismatch order from 5'-GA-3' to 5'-AG-3' results in an oligomer that does not form a duplex. These results agree with predictions from the model derived from NMR and molecular mechanics and indicate that the sequence 5'-pyrimidine-GA-purine-3' forms a stable conformational unit that fits quite well into a B-form double helix.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nearest-neighbor thermodynamics and NMR of DNA sequences with internal A.A, C.C, G.G, and T.T mismatches

Thermodynamic measurements are reported for 51 DNA duplexes with A.A, C.C, G.G, and T.T single mismatches in all possible Watson-Crick contexts. These measurements were used to test the applicability of the nearest-neighbor model and to calculate the 16 unique nearest-neighbor parameters for the 4 single like with like base mismatches next to a Watson-Crick pair. The observed trend in stabilities of mismatches at 37 degrees C is G.G > T.T approximately A.A > C.C. The observed stability trend for the closing Watson-Crick pair on the 5' side of the mismatch is G.C >/= C.G >/= A.T >/= T.A. The mismatch contribution to duplex stability ranges from -2.22 kcal/mol for GGC.GGC to +2.66 kcal/mol for ACT.ACT. The mismatch nearest-neighbor parameters predict the measured thermodynamics with average deviations of DeltaG degrees 37 = 3.3%, DeltaH degrees = 7. 4%, DeltaS degrees = 8.1%, and TM = 1.1 degrees C. The imino proton region of 1-D NMR spectra shows that G.G and T.T mismatches form hydrogen-bonded structures that vary depending on the Watson-Crick context. The data reported here combined with our previous work provide for the first time a complete set of thermodynamic parameters for molecular recognition of DNA by DNA with or without single internal mismatches. The results are useful for primer design and understanding the mechanism of triplet repeat diseases.     

Stability and mismatch discrimination of locked nucleic acid-DNA duplexes

Locked nucleic acids (LNA; symbols of bases, +A, +C, +G, and +T) are introduced into chemically synthesized oligonucleotides to increase duplex stability and specificity. To understand these effects, we have determined thermodynamic parameters of consecutive LNA nucleotides. We present guidelines for the design of LNA oligonucleotides and introduce free online software that predicts the stability of any LNA duplex oligomer. Thermodynamic analysis shows that the single strand-duplex transition is characterized by a favorable enthalpic change and by an unfavorable loss of entropy. A single LNA modification confines the local conformation of nucleotides, causing a smaller, less unfavorable entropic loss when the single strand is restricted to the rigid duplex structure. Additional LNAs adjacent to the initial modification appear to enhance stacking and H-bonding interactions because they increase the enthalpic contributions to duplex stabilization. New nearest-neighbor parameters correctly forecast the positive and negative effects of LNAs on mismatch discrimination. Specificity is enhanced in a majority of sequences and is dependent on mismatch type and adjacent base pairs; the largest discriminatory boost occurs for the central +C·C mismatch within the +T+C+C sequence and the +A·G mismatch within the +T+A+G sequence. LNAs do not affect specificity in some sequences and even impair it for many +G·T and +C·A mismatches. The level of mismatch discrimination decreases the most for the central +G·T mismatch within the +G+G+C sequence and the +C·A mismatch within the +G+C+G sequence. We hypothesize that these discrimination changes are not unique features of LNAs but originate from the shift of the duplex conformation from B-form to A-form.     

Thermodynamics of RNA hairpins containing single internal mismatches.

Thermodynamic parameters and circular dichroism spectra are presented for RNA hairpins containing single internal mismatches in the stem regions. Three different sequence contexts for the G*U mismatch and two contexts for C*A, G*A, U*U, A*C and U*G mismatches were examined and compared with Watson-Crick base-pair stabilities. The RNA hairpins employed were a microhelix and tetraloop representing the Escherichia coli tRNAAlaacceptor stem and sequence variants that have been altered at the naturally occurring G*U mismatch site. UV melting studies were carried out under different conditions to evaluate the effects of sodium ion concentration and pH on the stability of mismatch-containing hairpins. Our main findings are that single internal mismatches exhibit a range of effects on hairpin stability. In these studies, the size and sequence of the loop and stem are shown to influence the overall stability of the RNA, and have a minor effect on the relative mismatch stabilities. The relationship of these results to RNA-ligand interactions involving mismatch base-pairs is discussed.     

Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies.

We investigated the effects of various primer-template mismatches on DNA amplification of an HIV-1 gag region by the polymerase chain reaction (PCR). Single internal mismatches had no significant effect on PCR product yield while those at the 3'-terminal base had varied effects. A:G, G:A, and C:C mismatches reduced overall PCR product yield about 100-fold, A:A mismatches about 20-fold. All other 3'-terminal mismatches were efficiently amplified, although the G:G mismatches appeared to be more sensitive to sequence context and dNTP concentrations than other mismatches. It should be noted that mismatches of T with either G, C, or T had a minimal effect on PCR product yield. Double mismatches within the last four bases of a primer-template duplex where one of the mismatches is at the 3' terminal nucleotide, in general, reduced PCR product yield dramatically. The presence of a mismatched T at the 3'-terminus, however, allowed significant amplification even when coupled with an adjacent mismatch. Furthermore, even two mismatched Ts at the 3'-terminus allowed efficient amplification.     

Solution conformation of a deoxynucleotide containing tandem G.A mismatched base pairs and 3'-overhanging ends in d(GTGAACTT)2.

We have used 31P and 1H NMR spectroscopy and circular dichroism to define the solution conformation of d(GTGAACTT)2 which contains tandem G.A mismatched base pairs and 3'-overhanging TT ends. Measurements of coupling constants and NOE intensities show that the sugar puckers of the nucleotides are predominantly in the south domain (i.e., near C2'-endo) and that the glycosidic torsion angles are anti. The sequential NOE intensities indicate the presence of a right-handed helix. Analysis of the 31P and 1H NMR spectra of the duplex shows that the tandem mismatch forms a block in which there are unusual backbone torsion angles (i.e., in the BII state), within an otherwise B-like structure. The chemical shift of the N1H of the mismatched guanosine and NOEs between the mismatched base pairs and their nearest neighbors are inconsistent with the imino pairing present in single A.G mismatches or in the X-ray structure of a tandem mismatch [Privé, G. G., et al. (1987) Science 238, 498-503] but the data are consistent with the amino pairing found by Li et al. (1991) [Li, Y., et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 26-30]. The strong base-base stacking both within the tandem G.A block and between the G.A mismatches and their other nearest neighbors offsets the intrinsic destabilizing effects of the mismatch. Further, the 3'-TT overhangs stack onto the ends of the helix and stabilize the duplex against fraying, which accounts for the observed increase in the melting temperature compared with the flush-ended duplex.     

Effect of LNA- and OMeN-modified oligonucleotide probes on the stability and discrimination of mismatched base pairs of duplexes

Locked nucleic acid (LNA) and 2'-O-methyl nucleotide (OMeN) are the most extensively studied nucleotide analogues. Although both LNA and OMeN are characterized by the C3'-endo sugar pucker conformation, which is dominant in A-form DNA and RNA nucleotides, they demonstrate different binding behaviours. Previous studies have focused attention on their properties of duplex stabilities, hybridization kinetics and resistance against nuclease digestion; however, their ability to discriminate mismatched hybridizations has been explored much less. In this study, LNA- and OMeN-modified oligonucleotide probes have been prepared and their effects on the DNA duplex stability have been examined: LNA modifications can enhance the duplex stability, whereas OMeN modifications reduce the duplex stability. Next, we studied how the LNA:DNA and OMeN:DNA mismatches reduced the duplex stability. Melting temperature measurement showed that different LNA:DNA or OMeN:DNA mismatches indeed influence the duplex stability differently. LNA purines can discriminate LNA:DNA mismatches more effectively than LNA pyrimidines as well as DNA nucleotides. Furthermore, we designed five LNA- and five OMeN-modified oligonucleotide probes to simulate realistic situations where target-probe duplexes contain a complementary LNA:DNA or OMeN:DNA base pairs and a DNA:DNA mismatch simultaneously. The measured collective effect showed that the duplex stability was enhanced by the complementary LNA:DNA base pair but decreased by the DNA:DNA mismatch in a position-dependent manner regardless of the chemical identity and position of the complementary LNA:DNA base pair. On the other hand, the OMeN-modified probes also showed that the duplex stability was reduced by both the OMeN modification and the OMeN:DNA mismatch in a position-dependent manner.     

Thermodynamics of internal C.T mismatches in DNA.

Thermodynamics of 23 oligonucleotides with internal single C.T mismatches were obtained by measuring UV absorbance as a function of temperature. Results from these 23 duplexes were combined with three measurements from the literature to derive nearest-neighbor thermodynamic parameters for seven linearly independent trimer sequences with internal C.T mismatches. The data show that the nearest-neighbor model is adequate for predicting thermodynamics of oligonucleotides with internal C.T with average deviations for Delta G degrees37, Delta H degrees, Delta S degrees and T m of 6.4%, 9.9%, 10.6%, and 1.9 degreesC respectively. C.T mismatches destabilize the duplex in all sequence contexts. The thermodynamic contribution of C. T mismatches to duplex stability varies weakly depending on the orientation of the mismatch and its context and ranges from +1.02 kcal/mol for GCG/CTC and CCG/GTC to +1.95 kcal/mol for TCC/ATG.     

Free energy contributions of G.U and other terminal mismatches to helix stability

Thermodynamic parameters of helix formation were measured spectroscopically for seven hexaribonucleotides containing a GC tetramer core and G.U or other terminal mismatches. The free energies of helix formation are compared with those for the tetramer core alone and with those for the hexamer with six Watson-Crick base pairs. In 1 M NaCl, at 37 degrees C, the free energy of a terminal G.U mismatch is about equal to that of the corresponding A.U pair. Although other terminal mismatches studied add between -1.0 and -1.6 kcal/mol to delta G0 37 for helix formation, all are less stable than the corresponding Watson-Crick pairs. Comparisons of the stability increments for terminal G.U mismatches and G.C pairs suggest when stacking is weak the additional hydrogen bond in the G.C pair adds roughly -1 kcal/mol to the favorable free energy of duplex formation.     

Thermodynamics-structure relationship of single mismatches in RNA/DNA duplexes

Thermodynamics of 66 RNA/DNA duplexes containing single mismatches were measured by UV melting methods. Stability enhancements for rG. dT mismatches were the largest of all mismatches examined here, while rU.dG mismatches were not as stable. The methyl group on C5 of thymine enhanced the stability by 0.12 approximately 0.53 kcal mol(-)(1) depending on the identity of adjacent Watson-Crick base pairs, whereas the 2'-hydroxyl group in ribouridine stabilized the duplex by approximately 0.6 kcal mol(-)(1) regardless of the adjacent base pairs. Stabilities induced by the methyl group in thymine, the 2'-hydroxyl group of ribouridine, and an nucleotide exchange at rG.dT and rU.dG mismatches were found to be independent of each other. The order for the mismatch stabilities is rG.dT > rU. dG approximately rG.dG > rA.dG approximately rG.dA approximately rA. dC > rA.dA approximately rU.dT approximately rU.dC > rC.dA approximately rC.dT, although the identity of the adjacent base pairs slightly altered the order. The pH dependence stability and structural changes were suggested for the rA.dG but not for rG.dA mismatches. Comparisons of trinucleotide stabilities for G.T and G.U pairs in RNA, DNA, and RNA/DNA duplexes indicate that stable RNA/DNA mismatches exhibit a stability similar to RNA mismatches while unstable RNA/DNA mismatches show a stability similar to that of DNA mismatches. These results would be useful for the design of antisense oligonucleotides.     

Effects of GA mismatches on the structure and thermodynamics of RNA internal loops

UV melting, CD and NMR studies indicate rGCGAGCG and rGCAGGCG from unusually stable duplexes of type a and b. The observed delta G degree 37's in 1 M NaCl are -6.7 and -6.3 kcal/mol, respectively. For the related duplex, c, delta G degree 37 is -4.2 kcal/mol. The predicted delta G degree 37 from nearest-neighbor parameters (formula; see text) for all three duplexes is -4.7 kcal/mol (Freier, S.M., Kierzek, R., Jaeger, J.A., Sugimoto, N., Caruthers, M.H., Neilson, T., & Turner, D.H. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 9373-9377). The results suggest a special interaction in the duplexes containing GA mismatches. Presumably, this is hydrogen bonding between G and A. While the thermodynamics for (rGCGAGCG)2 and (rGCAGGCG)2 are similar, CD and the imino region of the proton NMR spectra indicate their structures are different. In particular, (rGCAGGCG)2 exhibits a CD spectrum typical of A-form geometry with a weak negative band at 280 nm. In contrast, the CD spectrum for (rGCGAGCG)2 has an intense positive band at 285 nm. The NMR spectrum of (rGCAGGCG)2 has a resonance corresponding to a hydrogen-bonded GA mismatch, while for (rGCGAGCG)2 no hydrogen-bonded imino proton is observed for the mismatch. The glycosidic torsion angles of the bases in the GA mismatches of (rGCAGGCG)2 and (rCGCAGGCG)2 are anti. Duplexes of type d, where X is A, G, or U, are more stable than e, and the stability differences are similar to those (formula; see text) observed for f versus g. Thus, 3'-dangling ends in this system make contributions to duplex stability that are similar to contributions observed with fully paired duplexes.     

Influence of nearest neighbor sequence on the stability of base pair mismatches in long DNA; determination by temperature-gradient gel electrophoresis.

Temperature-gradient gel electrophoresis (TGGE) was employed to determine the thermal stabilities of 48 DNA fragments that differ by single base pair mismatches. The approach provides a rapid way for studying how specific base mismatches effect the stability of a long DNA fragment. Homologous 373 bp DNA fragments differing by single base pair substitutions in their first melting domain were employed. Heteroduplexes were formed by melting and reannealing pairs of DNAs, one of which was 32P-labeled on its 5'-end. Product DNAs were separated based on their thermal stability by parallel and perpendicular temperature-gradient gel electrophoresis. The order of stability was determined for all common base pairs and mismatched bases in four different nearest neighbor environments; d(GXT).d(AYC), d(GXG).d(CYC), d(CXA).d(TYG), and d(TXT).d(AYA) with X,Y = A, T, C, or G. DNA fragments containing a single mismatch were destabilized by 1 to 5 degrees C with respect to homologous DNAs with complete Watson-Crick base pairing. Both the bases at the mismatch site and neighboring stacking interactions influence the destabilization caused by a mismatch. G.T, G.G and G.A mismatches were always among the most stable mismatches for all nearest neighbor environments examined. Purine.purine mismatches were generally more stable than pyrimidine.pyrimidine mispairs. Our results are in very good agreement with data where available from solution studies of short DNA oligomers.     

O6-methylguanine and A.C and G.T mismatches cause asymmetric structural defects in DNA that are affected by DNA sequence

Mismatched and modified base pairs are central to questions of DNA mutation and repair. NMR and X-ray crystallography of mispairs indicate little to no local helical distortion, but these techniques are not sensitive to more global distortions of the DNA molecule. We used polyacrylamide gel electrophoresis and thermal denaturation to examine A.C, G.T, and O6-methylG.T and O6-methylG.C mismatches synthesized in place of either of two adjacent G.C base pairs in synthetic DNA duplexes. Substitution for G.C at either position decreased the stability of the duplex; O6-methylguanine was more destabilizing in place of the 5'G than in place of the 3'G. Comparisons between polymers synthesized so that lesions occurred regularly spaced on the same side of the helix and polymers synthesized so that the lesions alternated from side to side on the helix showed that these lesions introduced helical distortion composed of (i) a symmetric frictional component, probably caused by localized bubble formation, and (ii) an asymmetric component indicative of a more global effect on the DNA molecule. Comparisons between these effects at the two adjacent positions show that the extent of structural perturbation depends on sequence context.     

Crystal structure and stability of a DNA duplex containing A(anti).G(syn) base-pairs

The synthetic dodecanucleotide d(CGCAAATTGGCG) has been analysed by single-crystal X-ray diffraction techniques and the structure refined to R = 0.16 and 2.25 A resolution, with the location of 94 solvent molecules. The sequence crystallizes as a full turn of a B-DNA helix with ten Watson-Crick base-pairs and two adenine-guanine mispairs. The analysis clearly shows that the mismatches are of the form A(anti).G(syn). Thermal denaturation studies indicate that the stability of the duplex is strongly pH dependent, with a maximum at pH 5.0, suggesting that the base-pair is stabilized by protonation. Three different arrangements have been observed for base-pairs between guanine and adenine and it is likely that A.G mismatch conformation is strongly influenced by dipole-dipole interactions with adjacent base-pairs.     

Crystal structure, stability and in vitro RNAi activity of oligoribonucleotides containing the ribo-difluorotoluyl nucleotide: insights into substrate requirements by the human RISC Ago2 enzyme

Short interfering RNA (siRNA) duplexes are currently being evaluated as antisense agents for gene silencing. Chemical modification of siRNAs is widely expected to be required for therapeutic applications in order to improve delivery, biostability and pharmacokinetic properties. Beyond potential improvements in the efficacy of oligoribonucleotides, chemical modification may also provide insight into the mechanism of mRNA downregulation mediated by the RNA–protein effector complexes (RNA-induced silencing complex or RISC). We have studied the in vitro activity in HeLa cells of siRNA duplexes against firefly luciferase with substitutions in the guide strand of U for the apolar ribo-2,4-difluorotoluyl nucleotide (rF) [Xia, J. et al. (2006) ACS Chem. Biol., 1, 176–183] as well as of C for rF. Whereas an internal rF:A pair adjacent to the Ago2 (‘slicer’ enzyme) cleavage site did not affect silencing relative to the native siRNA duplex, the rF:G pair and other mismatches such as A:G or A:A were not tolerated. The crystal structure at atomic resolution determined for an RNA dodecamer duplex with rF opposite G manifests only minor deviations between the geometries of rF:G and the native U:G wobble pair. This is in contrast to the previously found, significant deviations between the geometries of rF:A and U:A pairs. Comparison between the structures of the RNA duplex containing rF:G and a new structure of an RNA with A:G mismatches with the structures of standard Watson–Crick pairs in canonical duplex RNA leads to the conclusion that local widening of the duplex formed by the siRNA guide strand and the targeted region of mRNA is the most likely reason for the intolerance of human Ago2 (hAgo2), the RISC endonuclease, toward internal mismatch pairs involving native or chemically modified RNA. Contrary to the influence of shape, the thermodynamic stabilities of siRNA duplexes with single rF:A, A:A, G:A or C:A (instead of U:A) or rF:G pairs (instead of C:G) show no obvious correlation with their activities. However, incorporation of three rF:A pairs into an siRNA duplex leads to loss of activity. Our structural and stability data also shed light on the role of organic fluorine as a hydrogen bond acceptor. Accordingly, UV melting (T_M) data, osmotic stress measurements, X-ray crystallography at atomic resolution and the results of semi-empirical calculations are all consistent with the existence of weak hydrogen bonds between fluorine and the H-N1(G) amino group in rF:G pairs of the investigated RNA dodecamers.     

DNA triple-helix formation at target sites containing duplex mismatches

We have studied the formation of DNA triple helices at target sites that contain mismatches in the duplex target. Fluorescence melting studies were used to examine a series of parallel triple helices that contain all 64 N.XZ triplet combinations at the centre (where N, X and Z are each of the four natural DNA bases in turn). Similar experiments were also performed with N=bis-amino-U (BAU) (for stable recognition of AT base pairs) and N=S (for recognition of TA inversions). We find that the introduction of a duplex mismatch destabilises the C+.GZ, T.AZ and G.TZ triplets. A similar effect is seen with BAU.AZ triplets. In contrast, other base combinations, based on non-standard triplets such as C.AZ, T.TZ, G.CZ and A.CZ are stabilised by the presence of a duplex mismatch. In each case S binds to sites containing duplex mismatches better than the corresponding Watson-Crick base pairs.     

Structures of mismatched base pairs in DNA and their recognition by the Escherichia coli mismatch repair system.

The Escherichia coli mismatch repair system does not recognize and/or repair all mismatched base pairs with equal efficiency: whereas transition mismatches (G X T and A X C) are well repaired, the repair of some transversion mismatches (e.g. A X G or C X T) appears to depend on their position in heteroduplex DNA of phage lambda. Undecamers were synthesized and annealed to form heteroduplexes with a single base-pair mismatch in the centre and with the five base pairs flanking each side corresponding to either repaired or unrepaired heteroduplexes of lambda DNA. Nuclear magnetic resonance (n.m.r.) studies show that a G X A mismatch gives rise to an equilibrium between fully helical and a looped-out structure. In the unrepaired G X A mismatch duplex the latter predominates, while the helical structure is predominant in the case of repaired G X A and G X T mismatches. It appears that the E. coli mismatch repair enzymes recognize and repair intrahelical mismatched bases, but not the extrahelical bases in the looped-out structures.     

Influence of pH on the conformation and stability of mismatch base-pairs in DNA

A series of self-complementary dodecanucleotide duplexes containing two symmetrically disposed mismatches have been studied by pH-dependent, ultraviolet light melting techniques. The results indicate that A.C, and C.C mismatches are strongly stabilized by protonation and that the degree of stabilization of the A.C mismatch depends greatly on the flanking bases. In one case, a duplex containing two A.C mismatches is more stable than the native sequence below pH 5.5. The G.A mismatch displays conformational flexibility, with a protonated G(syn).A(anti) base-pair occurring in certain base stacking environments but not in others. The A.A and T.C mismatches are not stabilized at low pH. These solution studies correlate well with predictions based on X-ray crystallographic data.     

Nucleic acid duplex stability: influence of base composition on cation effects.

The effects of counter ion on a nucleic acid duplex stability were investigated. Since a linear free energy relationship for the thermostability of oligonucleotide duplexes between those in 1 M and in 100 mM NaCl-phosphate buffer were observed regardless of whether they are DNA-DNA, RNA-RNA or RNA-DNA duplexes, simple prediction systems for [Delta] G degrees 37as well as T mvalues in 100 mM NaCl-phosphate buffer were established. These predictions were successful with an average error of only 2.4 degrees C for T mand 5. 7% for G degrees 37values. The number of Na+newly bound to a duplex when the duplex forms (-[Delta] n) was significantly influenced by the base composition, and -[Delta] n for d(GCCAGTTAA)/d(TTAACTGGC) was different for MgCl2, CaCl2, BaCl2and MnCl2(from 0.70 to 0.76 with the same order of the duplex stability). Almost no additive effects on the duplex stability was observed for NaCl and MgCl2, suggesting a competitive binding for these cations. The sequence-dependent manner of [Delta] n suggests the presence of preferential base pairs or nearest-neighbor base pairs for the cation binding, which would affect nearest-neighbor parameters.     

Effect of the 1-(2'-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole residue on the stability of DNA duplexes and triplexes.

3-Nitropyrrole (M) was introduced as a non-discriminating 'universal' base in nucleic acid duplexes by virtue of small size and a presumed tendency to stack but not hydrogen bond with canonical bases. However, the absence of thermally-induced hyperchromic changes by single-stranded deoxyoligomers in which M alternates with A or C residues shows that M does not stack strongly with A or C nearest neighbors. Yet, the insertion of a centrally located M opposite any canonical base in a duplex is sometimes even less destabilizing than that of some mismatches, and the variation in duplex stability is small. In triplexes, on the other hand, an M residue centrally located in the third strand reduces triplex stability drastically even when the X.Y target base pair is A.T or G. C in a homopurine. homopyrimidine segment. But, when the target duplex opposition is M-T and the third strand residue is T, the presence of M in the test triplet has little effect on triplex stability. Therefore, a lack of hydrogen bonding in an otherwise helix-compatible test triplet cannot be responsible for triplex destabilization when M is the third strand residue. Thus, M is non-discriminating and none-too-destabilizing in a duplex, but in a triplex it is extremely destabilizing when in the third strand.