The 6-thioguanine/5-methyl-2-pyrimidinone base pair.

As part of a program to determine the physical possibility of expanding the number of types of base pairs in DNA, the pairing stabilities of the analog bases 6-thioguanine (GS) and 5-methyl-2-pyrimidinone (TH) in oligodeoxynucleotides were measured. Procedures were developed to synthesize oligodeoxynucleotides with the analog bases. The sequences of the synthesized oligomers were T-C-G-A-C-G-G-X-Y-C-C-G. An enzymatic procedure was developed to measure relative association constants of oligomer pairs with the self complementary reference oligomer, X = A and Y = T, K(T/A) = K. The results were K(C/G) = (5 +/- .5)K, K(TH/GS) = K/(1 +/- .5), K(T/G) = K/(9 +/- 3), K(TH/G) = K/(25 +/- 5), K(C/GS) less than K/30, K(TH/A) less than K/40, K(T/GS) less than K/40, K(C/A) less than K/40. The results with the standard bases are consistent with other methods of measurement. The stability of the base pair GS/TH is approximately the same as the standard base pair A/T.     

Synthesis and duplex stability of oligonucleotides containing cytosine-thymine analogues.

The synthesis of the deoxynucleoside derived from the base P, 6H,8H-3,4-dihydro-pyrimido[4,5-c] [4,5-c] [1,2]oxazin-7-one, 2, and its introduction by established phosphoramidite and H-phosphonate chemistry into oligonucleotides is described. The melting transition temperatures (Tm) of a range of heptadecamer duplexes containing P/A and P/G base-pairs are compared with corresponding ones having N4-methoxycytosine (M) 1 and mismatched normal bases. P/A and P/G pairs allow closely similar duplex stabilities and have the potential to reduce the multiplicity of probes and primers based on amino acid sequences by removing the T/C degeneracy.     

Nucleotide analogs and new buffers improve a generalized method to enrich for low abundance mutations

A high sensitivity method for detecting low level mutations is under development. A PCR reaction is performed in which a restriction site is introduced in wild-type DNA by alteration of specific bases. Digestion of wild-type DNA by the cognate restriction endonuclease (RE) enriches for products with mutations within the recognition site. After reamplification, mutations are identified by a ligation detection reaction (LDR). This PCR/RE/LDR assay was initially used to detect PCR error in known wild-type samples. PCR error was measured in low |Deltap K a| buffers containing tricine, EPPS and citrate, as well as otherwise identical buffers containing Tris. PCR conditions were optimized to minimize PCR error using perfect match primers at the Msp I site in the p53 tumor suppressor gene at codon 248. However, since mutations do not always occur within pre-existing restriction sites, a generalized PCR/RE/LDR method requires the introduction of a new restriction site. In principle, PCR with mismatch primers can alter specific bases in a sequence and generate a new restriction site. However, extension from 3' mismatch primers may generate misextension products. We tested conversion of the Msp I (CCGG) site to a Taq I site (TCGA). Conversion was unsuccessful using a natural base T mismatch primer set. Conversion was successful when modified primers containing the 6 H,8 H -3, 4-dihydropyrimido[4,5- c ][1,2]oxazine-7-one (Q6) base at 3'-ends were used in three cycles of preconversion PCR prior to conversion PCR using the 3' natural base T primers. The ability of the pyrimidine analog Q6 to access both a T-like and C-like tautomer appears to greatly facilitate the conversion.     

Base pairing involving deoxyinosine: implications for probe design.

The thermal stability of oligodeoxyribonucleotide duplexes containing deoxyinosine (I) residues matched with each of the four normal DNA bases were determined by optical melting techniques. The duplexes containing at least one I were obtained by mixing equimolar amounts of an oligonucleotide of sequence dCA3XA3G with one of sequence dCT3YT3G where X and Y were A, C, G, T, or I. Comparison of optical melting curves yielded relative stabilities for the I-containing standard base pairs in an otherwise identical base-pair sequence. I:C pairs were found to be less stable than A:T pairs in these duplexes. Large neighboring-base effects upon stability were observed. For example, when (X,Y) = (I,A), the duplex is eight-fold more stable than when (X,Y) = (A,I). Independent of sequence effects the order of stabilities is: I:C greater than I:A greater than I:T congruent to I:G. This order differs from that of deoxyguanosine which pairs less strongly with dA; otherwise each deoxyinosine base pair is less stable than its deoxyguanosine counterpart in the same sequence environment. Implications of these results for design of DNA oligonucleotide probes are discussed.     

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.     

Sequence dependent electrophoretic mobilities and melting temperatures for A-T containing oligodeoxyribonucleotides.

The electrophoretic mobilities and thermal melting properties of self complementary A-T containing dodecamer oligodeoxyribonucleotides have been investigated as a function of solution conditions. The oligomers contained tracts of nonalternating A-T base pairs of 2 (d(A2T2)3), 3 (d(A3T3)2), and 6 (d(A6T6] as well as the fully alternating (d(A-T)6) sequence. The melting temperature increased with the length of the nonalternating sequence and was approximately 12 degrees C higher in the d(A6T6) sequence than in the alternating oligomer. Under denaturing conditions all oligomers had the same electrophoretic mobility on acrylamide gels. Under conditions which favor duplex formation, the oligomers exhibited significant sequence dependent mobility differences. The mobilities of two oligomers, d(A-T)6 and d(A6-T6), were approximately equal and were less than those of the other oligonucleotides. The greatest mobility was observed for d(A2T2)3. These results are best explained by a model which requires bending at a junction of two or more continuous A or T bases with another sequence.     

Nucleotide analogs facilitate base conversion with 3' mismatch primers

We compared the efficiency of PCR amplification using primers containing either a nucleotide analog or a mismatch at the 3' base. To determine the distribution of bases inserted opposite eight different analogs, 3' analog primers were used to amplify four different templates. The products from the reactions with the highest amplification efficiency were sequenced.Analogs allowing efficient amplification followed by insertion of a new base at that position are herein termed 'convertides'. The three convertides with the highest amplification efficiency were used to convert sequences containing C, T, G and A bases into products containing the respective three remaining bases. Nine templates were used to generate conversion products, as well as non-conversion control products with no base change. We compared the ability of natural bases to convert specific sites with and without a preconversion step using nucleotide analog primers. Conversion products were identified by a ligation detection reaction using primers specific for the converted sequence. We found that conversions resulting in transitions were easier to accomplish than transversions and that sequence context influences conversion. Specifically, primer slippage appears to be an important mechanism for producing artifacts via polymerase extension of a 3' base or analog transiently base paired to neighboring bases of the template. Nucleotide analogs could often reduce conversion artifacts and increase the yield of the expected product. While new analogs are needed to reliably achieve transversions, the current set have proven effective for creating transition conversions.     

Binding of actinomycin D to single-stranded DNA of sequence motifs d(TGTCT(n)G) and d(TGT(n)GTCT)

Our recent binding studies with oligomers derived from base replacements on d(CGTCGTCG) had led to the finding that actinomycin D (ACTD) binds strongly to d(TGTCATTG) of apparent single-stranded conformation without GpC sequence. A fold-back binding model was speculated in which the planar phenoxazone inserts at the GTC site with a loop-out T base whereas the G base at the 3'-terminus folds back to form a basepair with the internal C and stacks on the opposite face of the chromophore. To provide a more concrete support for such a model, ACTD equilibrium binding studies were carried out and the results are reported herein on oligomers of sequence motifs d(TGTCT(n)G) and d(TGT(n)GTC). These oligomers are not expected to form dimeric duplexes and contain no canonical GpC sequences. It was found that ACTD binds strongly to d(TGTCTTTTG), d(TGTTTTGTC), and d(TGTTTTTGTC), all exhibiting 1:1 drug/strand binding stoichiometry. The fold-back binding model with displaced T base is further supported by the finding that appending TC and TCA at the 3'-terminus of d(TGTCTTTTG) results in oligomers that exhibit enhanced ACTD affinities, consequence of the added basepairing to facilitate the hairpin formation of d(TGTCTTTTGTC) and d(TGTCTTTTGTCA) in stabilizing the GTC/GTC binding site for juxtaposing the two G bases for easy stacking on both faces of the phenoxazone chromophore. Further support comes from the observation of considerable reduction in ACTD affinity when GTC is replaced by GTTC in an oligomer, in line with the reasoning that displacing two T bases to form a bulge for ACTD binding is more difficult than displacing a single base. Based on the elucidated binding principle of phenoxazone ring requiring its opposite faces to be stacked by the 3'-sides of two G bases for tight ACTD binding, several oligonucleotide sequences have been designed and found to bind well.     

Base sequence dependence of in vitro translesional DNA replication past a bulky lesion catalyzed by the exo- Klenow fragment of Pol I

The effects of base sequence, specifically different pyrimidines flanking a bulky DNA adduct, on translesional synthesis in vitro catalyzed by the Klenow fragment of Escherichia coli Pol I (exo(-)) was investigated. The bulky lesion was derived from the binding of a benzo[a]pyrene diol epoxide isomer [(+)-anti-BPDE] to N(2)-guanine (G*). Four different 43-base long oligonucleotide templates were constructed with G* at a site 19 bases from the 5'-end. All bases were identical, except for the pyrimidines, X or Y, flanking G* (sequence context 5'-.XGY., with X, Y = C and/or T). In all cases, the adduct G* slows primer extension beyond G* more than it slows the insertion of a dNTP opposite G* (A and G were predominantly inserted opposite G, with A > G). Depending on X or Y, full lesion bypass differed by factors of approximately 1.5-5 ( approximately 0.6-3.0% bypass efficiencies). A downstream T flanking G on the 5'-side instead of C favors full lesion bypass, while an upstream C flanking G* is more favorable than a T. Various deletion products resulting from misaligned template-primer intermediates are particularly dominant ( approximately 5.0-6.0% efficiencies) with an upstream flanking C, while a 3'-flanking T lowers the levels of deletion products ( approximately 0.5-2.5% efficiencies). The kinetics of (1) single dNTP insertion opposite G* and (2) extension of the primer beyond G* by a single dNTP, or in the presence of all four dNTPs, with different 3'-terminal primer bases (Z) opposite G* were investigated. Unusually efficient primer extension efficiencies beyond the adduct (approaching approximately 90%) was found with Z = T in the case of sequences with 3'-flanking upstream C rather than T. These effects are traced to misaligned slipped frameshift intermediates arising from the pairing of pairs of downstream template base sequences (up to 4-6 bases from G*) with the 3'-terminal primer base and its 5'-flanking base. The latter depend on the base Y and on the base preferentially inserted opposite the adduct. Thus, downstream template sequences as well as the bases flanking G* influence DNA translesion synthesis.     

Effects of bulge composition and flanking sequence on the kinking of DNA by bulged bases

We recently showed that bulged bases kink duplex DNA, with the degree of kinking increasing in roughly equal increments as the number of bases in the bulge increases from one to four [Hsieh, C.-H., & Griffith, J.D. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4833-4837]. Here we have examined the kinking of DNA by single A, C, G, or T bulges with different neighboring base pairs. Synthetic 30 base pair (bp) duplex DNAs containing 2 single-base bulges spaced by 10 bp were ligated head to tail, and their electrophoretic behavior in highly cross-linked gels was examined. All bulge-containing DNAs showed marked electrophoretic retardations as compared to non-bulge-containing DNA. Regardless of the sequence of the flanking base pairs, purine bulges produced greater retardations than pyrimidine bulges. Furthermore, C and T bulges produced the same retardations as did G and A bulges. Bulged DNA containing different flanking base pairs showed marked differences in electrophoretic mobility. For C-bulged DNA, the greatest retardations were observed with G.C neighbors, the least with T.A neighbors, and an intermediate amount with a mixture of neighboring base pairs. For A-bulged DNA, the retardations were greatest with G.C neighbors, less with T.A neighbors, even less with a mixture of neighboring base pairs, and finally least with C.G neighbors. Thus flanking base pairs affect C-bulged DNA and A-bulged DNA differently, and G.C and C.G flanking base pairs were seen to have very different effects. These results imply an important role of base stacking in determining how neighboring base pairs influence the kinking of DNA by a single-base bulge.     

Biochemical behavior of N-oxidized cytosine and adenine bases in DNA polymerase-mediated primer extension reactions

To clarify the biochemical behavior of 2'-deoxyribonucleoside 5'-triphosphates and oligodeoxyribonucleotides (ODNs) containing cytosine N-oxide (C(o)) and adenine N-oxide (A(o)), we examined their base recognition ability in DNA duplex formation using melting temperature (T(m)) experiments and their substrate specificity in DNA polymerase-mediated replication. As the result, it was found that the T(m) values of modified DNA-DNA duplexes incorporating 2'-deoxyribonucleoside N-oxide derivatives significantly decreased compared with those of the unmodified duplexes. However, single insertion reactions by DNA polymerases of Klenow fragment (KF) (exo(-)) and Vent (exo(-)) suggested that C(o) and A(o) selectively recognized G and T, respectively. Meanwhile, the kinetic study showed that the incorporation efficiencies of the modified bases were lower than those of natural bases. Ab initio calculations suggest that these modified bases can form the stable base pairs with the original complementary bases. These results indicate that the modified bases usually recognize the original bases as partners for base pairing, except for misrecognition of dATP by the action of KF (exo(-)) toward A(o) on the template, and the primers could be extended on the template DNA. When they misrecognized wrong bases, the chain could not be elongated so that the modified base served as the chain terminator.     

Bacteriophage T4 DNA primase-helicase. Characterization of the DNA synthesis primed by T4 61 protein in the absence of T4 41 protein

The bacteriophage T4 61/41 protein primase-helicase is part of a seven T4 protein system needed for DNA synthesis in vitro. Although both 41 and 61 proteins are required for the synthesis and utilization of the normal pppApC(pN)3 pentanucleotide primer, we show in the accompanying paper (Hinton, D. M., and Nossal, N. G. (1987) J. Biol. Chem. 262, 10873-10878) that high concentrations of 61 protein alone carry out a limited, template-dependent oligonucleotide synthesis with the dimers pppApC and pppGpC as the major products labeled with [alpha-32P]CTP. At these high concentrations, 61 protein alone primes DNA synthesis by T4 DNA polymerase and the T4 genes 44/62 and 45 polymerase accessory proteins, or by Escherichia coli DNA polymerase I. The addition of T4 replication proteins other than 41 protein does not change the size distribution of oligonucleotides made by 61 protein. However, the primers used for DNA synthesis in the absence of 41 protein are not dimers, but rather trace quantities of longer oligonucleotides (5 to about 45 bases) which begin predominantly with pppGpC. These results show that 41 protein is required to prime with oligonucleotides beginning with pppApC and suggest that 41 protein, either alone or in conjunction with 61 protein, helps to stabilize the usual short pentamer primers on the template until they are elongated by the DNA polymerase. Moreover, since 61 protein by itself can only initiate DNA synthesis with primers beginning with pppGpC, but cannot make oligonucleotides starting with pppGpC on T4 DNA in which all the C is glucosylated and hydroxymethylated, both the T4 41 and 61 proteins are essential to prime DNA synthesis on their normal template. In our analysis of RNA-primed DNA, we demonstrate that although RNA primers at the 5' ends of DNA chains are relatively resistant to the 3' to 5' exonuclease of T4 DNA polymerase (Kurosawa, Y., and Okazaki, T. (1979) J. Mol. Biol. 135, 841-861), pppNpNpNpNpN oligomers are digested to a greater extent than the dephosphorylated pentamers NpNpNpNpN.     

Factors that contribute to efficient catalytic activity of a small Ca2+-dependent deoxyribozyme in relation to its RNA cleavage function

Recently, we found a small Ca(2+)-dependent deoxyribozyme (unmodified), d(GCCTGGCAG(1)G(2)C(3)T(4)A(5)C(6)A(7)A(8)C(9)G(10)A(11)GTCCCT), with cleavage activity for its RNA substrate, r(AGGGACA downward arrow UGCCAGGC) ( downward arrow denotes the RNA cleavage site), in the presence of Ca(2+) and developed a functional SPR sensor chip with this deoxyribozyme [Okumoto, Y., Ohmichi, T., and Sugimoto, N. (2002) Biochemistry 41, 2769-2773]. In the study presented here, to clarify the factors contributing to the efficient catalytic activity of the unmodified deoxyribozyme, RNA cleavage reactions were carried out using 24 mutant deoxyribozymes containing one unnatural DNA nucleotide, such as dI (2'-deoxyinosine), 7-deaza-dG, 2-aminopurine, 7-deaza-dA, 2-amino-dA, dm(5)C (5-methyl-2'-deoxycytosine), or d(P)C (5-propynyl-2'-deoxycytosine). The K(m) values (Michaelis constants) with the mutants that lacked N7 and O6 of G(1) and O6 of G(2) were 4.5 and 6.6 times that of the unmodified one, respectively. The k(cat) value (cleavage rate constant) with the mutants that lacked O6 of G(10) was 0.025 times that of the unmodified one. The results of UV melting curves, SPR kinetics, and CD spectra supported the quantitative idea that the catalytic activity of the unmodified form was achieved using Ca(2+). On the basis of these results, a preliminary model for two G(1) x A(8) and G(2) x A(7) mismatched base pairs such as G(anti) x A(anti) formed in the catalytic loop is proposed. The factor of 10 increase in the k(cat)/K(m) value of the mutant deoxyribozyme, which has C(9) substituted with d(P)C, suggests that the base stacking interaction between the substituted propynyl group in dC and the nearest-neighbor base grew stronger. Thus, substituting d(P)C for dC in the catalytic loop would be one of the best ways to increase the catalytic activity of the deoxyribozyme.     

Unusual duplex formation in purine rich oligodeoxyribonucleotides

The purine rich oligodeoxyribonucleotides 1C, d(ATGACGGAATA) and 2C, d(ATGAGCGAATA) alone exhibit highly cooperative melting transitions. Analysis of the concentration dependence of melting, and electrophoretic studies indicate that these oligomers can form an unusual purine rich offset double helix. The unusual duplex is predicted to contain four A.T, two G.C, and four G.A mismatch base pairs as well as a single A base stacked on the 3' end of each chain of the helix. Other possible models for the duplex are unlikely because they are predicted to contain many base pairs of low stability. Changing the central sequence to CGG or GGG should destabilize the duplex and this is observed. The unusual duplex of 2C is more stable than the duplex of 1C indicating that the stability of G.A base pairs is quite sensitive to the surrounding sequence. Addition of 1C and 2C to their complementary pyrimidine strands results in normal duplexes of similar stability. We feel that the unusual duplexes are significantly stabilized by the intrinsic stacking tendency of purine bases.     

The ordering of nucleotides in the DNA: strong pyrimidine-purine patterns near homooligomer tracts

Here, we study the frequencies of occurrence of homooligomers flanked by one base, XnU or UXn, where X = A, C, G, T and U not equal to X. Specifically, we search for preferences (or discriminations) in their nearest neighbor doublet, VV. Extensive analysis of the data base reveals striking patterns in such VVUXn or UXn VV oligomers (V = A, C, G, T). With very few exceptions, if the VV and Xn are composed of complementary nucleotides, those oligomers having a pyrimidine (Y)-purine (R) junction are preferred over those with an RY one. If the VV and Xn nucleotides are not complementary, the RY junction oligomers are preferred over their YR counterparts. These trends are observed consistently in eukaryotic and prokaryotic sequences. They are particularly striking in the YR greater than RY oligomers containing complementary nucleotides. The general preferences and discriminations described here are in the same direction as our previous results for homooligomer tracts. These recurrences, along with some additional universal "rules", aid in our understanding of the ordering of nucleotides in the DNA.     

Energy barriers and rates of tautomeric transitions in DNA bases: ab initio quantum chemical study

Tautomeric transitions of DNA bases are proton transfer reactions, which are important in biology. These reactions are involved in spontaneous point mutations of the genetic material. In the present study, intrinsic reaction coordinates (IRC) analyses through ab initio quantum chemical calculations have been carried out for the individual DNA bases A, T, G, C and also A:T and G:C base pairs to estimate the kinetic and thermodynamic barriers using MP2/6-31G** method for tautomeric transitions. Relatively higher values of kinetic barriers (about 50-60 kcal/mol) have been observed for the single bases, indicating that tautomeric alterations of isolated single bases are quite unlikely. On the other hand, relatively lower values of the kinetic barriers (about 20-25 kcal/mol) for the DNA base pairs A:T and G:C clearly suggest that the tautomeric shifts are much more favorable in DNA base pairs than in isolated single bases. The unusual base pairing A':C, T':G, C':A or G':T in the daughter DNA molecule, resulting from a parent DNA molecule with tautomeric shifts, is found to be stable enough to result in a mutation. The transition rate constants for the single DNA bases in addition to the base pairs are also calculated by computing the free energy differences between the transition states and the reactants.     

The point mutation induced by the low-energy N+ ion implantation in Impatiens balsamine genome

To investigate the effect of genome mutations induced by low energy ions implantation in higher plants, genome mutation of Impatiens balsamine mutant induced by low energy N⁺ ion implantation were analyzed by the RAPD, ISSR and genome sequence. Six out of the 121 ISSR primers and 6 out of the 135 RAPD primers showed that polymorphism ratios between mutants and wild type were 4.96% and 2.89%, respectively. Sequence analysis revealed that base deletions, insertions, and substitutions were observed in the mutant genome comparable to wild type. N+ induced point mutations were mostly base substitution (77.4%), no duplication, long fragments insertions and deletions was found. In all point mutation, adenine (A) was most sensitive to the N⁺ ion implantation in impatiens. The transition was mainly A ?? guanine (G) (15.90%) and thymine (T) cytosine (C) (12.55%). Transversion happened in A ? T (16.74%), which much higher than C ? G (5.02%), G ? T (6.69%), A ? C (7.11%) bases. These findings indicate that low energy ions being a useful mutagen were mostly cause the point mutation in impatiens.     

Structural studies of DNA fragments: the G.T wobble base pair in A, B and Z DNA; the G.A base pair in B-DNA

The crystal structures of five double helical DNA fragments containing non-Watson-Crick complementary base pairs are reviewed. They comprise four fragments containing G.T base pairs: two deoxyoctamers d(GGGGCTCC) and d(GGGGTCCC) which crystallise as A type helices; a deoxydodecamer d(CGCGAATTTGCG) which crystallises in the B-DNA conformation; and the deoxyhexamer d(TGCGCG), which crystallises as a Z-DNA helix. In all four duplexes the G and T bases form wobble base pairs, with bases in the major tautomer forms and hydrogen bonds linking N1 of G with O2 of T and O6 of G with N3 of T. The X-ray analyses establish that the G.T wobble base pair can be accommodated in the A, B or Z double helix with minimal distortion of the global conformation. There are, however, changes in base stacking in the neighbourhood of the mismatched bases. The fifth structure, d(CGCGAATTAGCG), contains the purine purine mismatch G.A where G is in the anti and A in the syn conformation. The results represent the first direct structure determinations of base pair mismatches in DNA fragments and are discussed in relation to the fidelity of replication and mismatch recognition.     

Thermodynamic analysis of stacking hybridization of oligonucleotides with DNA template.

Contiguous stacking hybridization of oligodeoxyribonucleotides with DNA as template was investigated using three types of complexes: oligonucleotide contiguously stacked with the stem of the preformed minihairpin (complexes I), oligonucleotide tandems containing two (complexes II) or three (complexes III) short oligomers with a common DNA template. Enthalpy Delta H degrees and entropy Delta S degrees of the coaxial stacking of adjacent duplexes were determined for GC/G*pC, GT/A*pC, AC/G*pT, AT/A*pT, CT/A*pG, AG/C*pT, AA/T*pT and TT/A*pA nicked (*) dinucleotide base pairs. The maximal efficiency of co-operative interaction was found for the GC/G*pC interface (Delta G degrees(NN/N*pN)=-2.7 kcal/mol) and the minimal one for the AA/T*pT interface (Delta G degrees(NN/N*pN)=-1.2 kcal/mol) at 37 degrees C. As a whole, the efficiency of the base pairs interaction Delta G degrees(NN/N*pN) in the nick is not lower than that within the intact DNA helix (Delta G degrees(NN/NN)).These observed Delta G degrees(NN/N*pN) values are proposed may include the effect of the partial removal of fraying at the adjacent helix ends additionally to the effect of the direct stacking of the terminal base pairs in the duplex junction (Delta G degrees(NN/NN). The thermodynamic parameters have been found to describe adequately the formation of all tandem complexes of the II and III types with oligonucleotides of various length and hybridization properties. The performed thermodynamic analysis reveals features of stacking oligonucleotide hybridization which allow one to predict the temperature dependence of association of oligonucleotides and the DNA template within tandem complexes as well as to determine optimal concentration for formation of these complexes characterized by high co-operativity level.