Identification of a genetically defined ultra-high-risk group in relapsed pediatric T-lymphoblastic leukemia.

In the search for genes that define critical steps of relapse in pediatric T-cell acute lymphoblastic leukemia (T-ALL) and can serve as prognostic markers, we performed targeted sequencing of 313 leukemia-related genes in 214 patients: 67 samples collected at the time of relapse and 147 at initial diagnosis. As relapse-specific genetic events, we identified activating mutations in NT5C2 (P=0.0001, Fisher's exact test), inactivation of TP53 (P=0.0007, Fisher's exact test) and duplication of chr17:q11.2-24.3 (P=0.0068, Fisher's exact test) in 32/67 of T-ALL relapse samples. Alterations of TP53 were frequently homozygous events, which significantly correlated with higher rates of copy number alterations in other genes compared with wild-type TP53 (P=0.0004, Mann-Whitney's test). We subsequently focused on mutations with prognostic impact and identified genes governing DNA integrity (TP53, n=8; USP7, n=4; MSH6, n=4), having key roles in the RAS signaling pathway (KRAS, NRAS, n=8), as well as IL7R (n=4) and CNOT3 (n=4) to be exclusively mutated in fatal relapses. These markers recognize 24/49 patients with a second event. In 17 of these patients with mostly refractory relapse and dire need for efficient treatment, we identified candidate targets for personalized therapy with p53 reactivating compounds, MEK inhibitors or JAK/STAT-inhibitors that may be incorporated in future treatment strategies.

Impaired speech perception in aphasic patients: event-related potential and neuropsychological assessment.

The mismatch negativity component (MMN) of auditory event-related potentials (ERP) was recorded in four aphasic patients and in age, gender and education matched controls. The MMN changes elicited by tone, vowel, voicing stop consonant and place-of articulation contrasts were recorded over both hemispheres. The results of MMN amplitude, latency and distribution differences between aphasics and controls were analyzed in detail. An extensive neuropsychological investigation was performed in order to highlight the assumed dissociation and possible interactions between the impaired acoustic/phonetic perception and deficient comprehension in aphasic patients. Our principal finding was that MMN elicited by pitch deviations is not enough sensitive to distinguish between patients and age-matched controls. The MMN elicited by consonant contrasts was found to be the most vulnerable in aphasic patients investigated. The MMN elicited by voicing ([ba:] vs. [pa:]) and place-of-articulation ([ba:] vs. [ga:]) could be characterized by total lack, distorted or very limited distribution and correlated with the patients' performance shown in the behavioral phoneme discrimination task. The magnitude of the deficient phoneme (vowel and consonant contrasts) processing shown by MMN anomalies was proportionally related to the non-word discrimination and did not interact with the word discrimination performance. The impact of deficient speech sound processing on higher level processes may depend on the type of aphasia, while the presence of perceptual deficits in processing acoustic/phonetic contrasts seems to be independent of the type of aphasia.

15-mer DNA duplexes containing an abasic site are thermodynamically more stable with adjacent purines than with pyrimidines.

Abasic site (AP)-containing duplexes, with flanking adenine (A) or cytosine (C) bases, were shown to be more stable with flanking A than with C bases [Sági, J., Hang, B., and Singer, B. (1999) Chem. Res. Toxicol. 12, 917-923]. We investigated whether the lower-magnitude destabilization by an AP site, with A neighbors, is a general effect of the purine versus the pyrimidine neighbors. Duplex stability, as compared to that of the corresponding control duplexes, was markedly decreased by the incorporation of the AP site (x) opposite any of the four bases. However, for the duplexes containing T, A, or C opposite the AP site, replacement of the symmetric doublet flanking pyrimidine bases with purines resulted in a smaller destabilization effect. The average stabilizing effect of the symmetric doublet purine neighbors of an AP site opposite T, A, or C bases was 3.2 degrees C (DeltaT(m)) and 1.3 kcal/mol (DeltaDeltaG degrees (37)) compared to those of pyrimidine neighbors. In contrast, a G.AP pair reduced or eliminated the differential effect of the neighbors. Using unrestrained molecular dynamics, it was shown that for the duplexes containing T opposite the AP site, with doublet pyrimidine neighbors, there was a larger magnitude of curvature around the lesion site than for the duplexes with the purines flanking the AP site. Purines flanking the AP site tend to shift toward each other, creating overlap, in contrast to the flanking pyrimidines. This indicates the possibility of stacking between purine bases at the AP site and can be the reason for the observed smaller thermodynamic destabilization of the duplexes with the AAxAA and GGxGG central sequences, as compared to those with TTxTT and CCxCC sequences. This work showed that for an AP site the GC content is not the only determinant of duplex stability, but rather is influenced more by whether purines or pyrimidines flank the AP site.

A structure-function study of nucleic acid-fluorenone complexes.

Several 2.7-bis-[(dialkylamino)-acetylamino]-fluoren-9-one derivatives (fluoramides) were synthesized as analogues of the DNA binding compound tilorone (2,7-bis[(diethylamino)-ethoxy]-fluoren-9-one). Previous studies showed the drugs to induce cytokines and inhibit reverse transcription. Here, their binding to DNA was evaluated using UV and circular dichroism studies. Like tilorone, the fluoramides derivatives also intercalate resulting in increased Tm values and new CD signatures. A preference to alternating A-T and G-C sequences was detected; only minor interaction to homologous sequences was observed. Moreover, no upper limit in the drug/DNA ratio was found, testing limit being the precipitation of the drug. However, surface plasmon resonance (SPR) studies of tilorone and 2,7-bis-[(dipropylamino)-acetyl-amino]-fluoren-9-one, indicate an astonishing drug/base pair ratio (r > 1), which point to a multitude of interactions under SPR conditions. Molecular modeling calculations, where the geometries of the complexes optimized under the assumption of intercalative and multitude of suprahelical arrangements, rationalize the observations. Based on the thermodynamic and biological studies, a structure-function model is proposed.

Differential destabilization of the DNA oligonucleotide double helix by a T.G mismatch, 3,N(4)-ethenocytosine, 3,N(4)-ethanocytosine, or an 8-(hydroxymethyl)-3,N(4)-ethenocytosine adduct incorporated into the same sequence contexts.

The T.G mismatch and the exocyclic adduct 3,N(4)-ethenocytosine (epsilonC) are repaired by the same enzyme, the human G/T(U) mismatch-DNA glycosylase (TDG). This enzyme removes the T, U, or epsilonC base from duplex DNA. The rate of cleavage was found to differ with the lesion and was also affected by neighbor sequences [Hang, B., Medina, M., Fraenkel-Conrat, H., and Singer, B. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 13561-13566]. Since sequence influences duplex stability, we determined the thermodynamic stability of T.G and epsilonC-containing 15-mer duplexes in which the bases flanking the lesion were systematically varied. The duplexes contained central 5'-TTXTT, 5'-AAXAA, 5'-CCXCC, or 5'-GGXGG sequences, where X is T, epsilonC, or two closely related structural derivatives of epsilonC: 3,N(4)-ethanocytosine (EC) and 8-(hydroxymethyl)-epsilonC (8-HM-epsilonC). Each of the four lesions, incorporated opposite G, decreased both the thermal (T(m)) and thermodynamic stability (DeltaG degrees (37)) of the 15-mer control duplexes. On the basis of the T(m) and DeltaG degrees (37) values, the order of destabilization of the TTXTT sequence in 15-mer duplexes was as follows: 8-HM-epsilonC > EC > epsilonC > T.G. The DeltaT(m) values range from -15.8 to -9.5 degrees C when C(t) = 8 microM. Duplexes with flanking AA or TT neighbors were more destabilized, by an average of 2 degrees C, than those with flanking GG or CC neighbors. The base opposite the modified base also influenced duplex stability. Within the TT context, of the four changed bases opposite the adducts, C had the greatest destabilizing effect, up to -18.4 degrees C. In contrast, a G opposite an adduct was generally the least destabilizing, and the smallest value was -3. 0 degrees C. Destabilizations were enthalpic in origin. Thus, this work shows that independently changing the modified base, the sequence, or the base opposite the lesion each affects the stability of the duplex, to significantly varying extents. The potential contribution of the thermodynamic stability to repair efficiency is discussed.

Sequence-dependent conformational perturbation in DNA duplexes containing an epsilonA.T mismatch using molecular dynamics simulation.

Previous experiments from this laboratory showed that 1, N:(6)-ethenoadenine (epsilonA) in 15mer DNA oligonucleotide duplexes with GGepsilonAGG and CCepsilonACC central sequences is repaired 3-5-fold more efficiently than in duplexes containing AAepsilonAAA and TTepsilonATT central sequences. This sequence dependence in repair rates appeared to correlate with the observed thermodynamic stability of these duplexes [Hang et al. (1998) J. Biol. Chem., 273, 33406-33413]. In the present work, unrestrained molecular dynamics was used to evaluate the sequence-dependent structural features of these duplexes. Explicit solvent and the particle mesh Ewald method were applied for the accurate representation of the electrostatic interactions. The differences observed in the axis- and intra-base pair parameters were primarily localized at the epsilonA*T mismatch in all sequences and indicate conformational diversity between the structures. However, all four structures remained in the B-conformational family. In the tip, tilt and propeller twist parameters for the five central base pairs, larger perturbations were found for the two duplexes with epsilonA flanked by A or T bases than for duplexes with epsilonA flanked by G or C bases. As a result of these perturbations, the average global curvature of the AAepsilonAAA and TTvarepsilonATT DNA duplexes was larger by approximately 12 degrees than that of the duplexes with the GGepsilonAGG and CCepsilonACC central sequences. The observed conformational differences between the duplexes containing A or T and G or C neighbors of epsilonA may contribute to the observed differential enzymatic repair of the same sequences.

Scalp distribution of the dimensional complexity of the EEG and the P3 ERP component in stroke patients.

The use of recently developed techniques allows the quantitative investigation of the non-linear properties of the electrical activity of the brain not only in basic but also in applied research. The point correlation dimension (PD2) was used in this study for the analysis of the electroencephalogram (EEG) recorded in patients with unilateral stroke caused by middle cerebral artery occlusion. The scalp distribution of the PD2 and that of the P3 event-related potential component was mapped and frequency spectra were calculated. Compared to normal controls, asymmetrical PD2 distribution was observed with low values on the side of the stroke, the extension of which depended on recording conditions (level of vigilance) and only partially corresponded to the region characterized by slow frequencies. In one case, ipsilateral reduction of the P3 wave was caused by a small subcortical stroke. The efficacy of the linear and non-linear methods in localizing brain pathology are evaluated and compared.

Sequence-dependent repair of synthetic AP sites in 15-mer and 35-mer oligonucleotides: role of thermodynamic stability imposed by neighbor bases.

We previously reported that 15-mer oligonucleotides with a central 1, N(6)-epsilonA were cleaved by alkylpurine-DNA N-glycosylase as a function of T(m), modulated by neighbor bases [Hang, B., Sági, J., and Singer, B. (1998) J. Biol. Chem. 273, 33406-33413]. This type of investigation has now been extended to cleavage by Escherichia coli endonuclease IV of a centrally placed synthetic AP site using both 15-mer and 35-mer duplexes. In 15-mers, the triplet sequences adjunct to the central AP site greatly affected the thermodynamic stability. The repair rate paralleled the thermal stability since endonuclease IV requires a double-stranded substrate. When the AP site-containing duplexes were 35-mers, there was also a general correlation between the thermostability and cleavage efficiency. However, the difference in the cleavage rates between different sequences was much less than with the 15-mers. Since the 35-mers were more than 96% annealed, this difference presumably results from local stability and structure adjacent to the AP site. These results suggest that under enzyme limiting conditions or overproduction of AP sites, sequence-dependent differential repair could occur in vivo.

Enzymatic hydrolysis and biological activity of oligonucleotides containing 5-substituted pyrimidine bases.

Two series of alternating ODNs containing 5-n.alkyl-, alkenyl- and alkynyl-dU and -dC units have been prepared in order to study the kinetics of their hydrolysis by SV PDE and human serum, respectively. Both in (r5dUpdA)10 and (r5dCpdG)6 series the rate of hydrolysis decreased with increasing length of side-chain. Replacement of thymidines by 5-hexynyl-dU in different antisense oligomers resulted in considerably higher biological activity relative to that of the thymidine-containing counterparts.

Thermal destabilization of DNA oligonucleotide duplexes by exocyclic adducts on adenine or cytosine depends on both the base and the size of adduct.

Numerous carcinogens or their bifunctional metabolites modify DNA bases by forming additional exocyclic rings on the base moiety. These modifications form exocyclic rings between N1 and N6 of dA, N3 and N4 of dC or N1 and N2 of dG, as well as the N2 and N3 of dG. This study focuses on the reaction products of dA and dC with chloroacetaldehyde, bis-chloroethyl nitrosourea and para-benzoquinone, which form etheno, ethano and para-benzoquinone derivatives, respectively. The three dC adducts and three dA adducts were each incorporated site-specifically into 25-nucleotide-long deoxyoligonucleotides. All duplexes with a single modified dA or dC adduct opposite the normal complement showed decreased thermal stability, as compared with the unmodified control duplex. The destabilizations ranges from -2 degrees C to -13 degrees C, depending on saturation, the size of the adduct and the nature of the base. Energy-minimized molecular models of the duplexes illustrate various degrees of distortions by the adducts, the para-benzoquinone adducts showing the greatest distortion.

Correlation between sequence-dependent glycosylase repair and the thermal stability of oligonucleotide duplexes containing 1, N6-ethenoadenine.

Previous experiments on DNA sequence context reported that base modification, replication, and repair are affected by the nature of neighbor bases. We now report that repair by mammalian alkylpurine-DNA-N-glycosylases (APNG) of 15-mer oligonucleotides with a central 1,N6-ethenoadenine (epsilonA), flanked by 5' and 3' tandem bases, is also highly sequence dependent. Oligonucleotides with the central sequences -GGepsilonAGG- or -CCepsilonACC- are repaired 3-5-fold more efficiently than those containing -AAepsilonAAA- or -TTepsilonATT- when using human or mouse APNG. Melting curves of the same duplexes showed that oligomers with G.C/C. G neighbors were less denatured than those with A.T/T.A neighbors at 37 degreesC. This sequence-dependent difference in denaturation correlates with the relative thermodynamic stability of oligomers with G.C/C.G or A.T/T.A neighbors. The dependence of repair on thermal stability was confirmed by enzyme reactions performed over 0-45 degreesC. Under these conditions, repair of epsilonA flanked by G.C/C.G was dramatically increased at 37 degreesC with continuous increase up to 45 degreesC, in contrast to that with flanking A.T/T. A pairs, which was in agreement with the degree of denaturation of these duplexes. These results indicate that the thermodynamic stability conferred by base pairs flanking epsilonA plays an essential role in maintaining the integrity of the duplex structure which is necessary for repair.

Differential cleavage of oligonucleotides containing the benzene-derived adduct, 1,N6-benzetheno-dA, by the major human AP endonuclease HAP1 and Escherichia coli exonuclease III and endonuclease IV.

We report here that the newly synthesized DNA adduct, 1,N6-benzetheno-dA (pBQ-dA), in defined oligonucleotides [Chenna and Singer, Chem. Res. Toxicol., 8, 865-874], is a substrate for the major human AP endonuclease, HAP1, and the Escherichia coli AP endonucleases, exonuclease III and endonuclease IV. The mechanism of cleavage is identical to that reported previously for 3,N4-benzetheno-dC (pBQ-dC) and leads to a phosphodiester bond cleavage 5' to the adduct. There are, however, significant differences in the rate of cleavage of this adduct by these enzymes. The two bacterial AP endonucleases are both much more efficient than the human repair enzyme. In addition, using two random oligodeoxynucleotide sequences containing a single pBQ-dA, exonuclease III and endonuclease IV are similarly active, while HAP1 shows a distinct sequence preference of approximately 10-fold in efficiency of cleavage. The repair of this adduct by the three recombinant enzymes is further confirmed by using both active site mutant HAP1 proteins and by E.coli mutant strains lacking exonuclease III and/ or endonuclease IV. This sequence-dependent repair of pBQ-dA by HAP1 may play an important role in modulating benzene-induced carcinogenesis.

A single cyclic p-benzoquinone adduct can destabilize a DNA oligonucleotide duplex.

p-Benzoquinone (p-BQ), a stable metabolite of the human carcinogen benzene, forms two-ring benzetheno exocyclic base adducts with C, A, and G bases in DNA. As a part of a project for studying the biological effect of the p-BQ adducts, we report here on the first biophysical characterization of oligodeoxyribonucleotide duplexes containing a single site-specific p-BQ-C, using thermal denaturation and circular dichroism (CD). We find that the thermal and thermodynamic stabilities of the control duplex are reduced by p-BQ-C. The Tm value decreases by 12.6 degrees C at the duplex concentration of 1.5 microM and the Delta G o by 10.2 kcal/mol. The latter was determined from the concentration dependence of the Tm values. The destabilization has little dependence on the nature of the opposite base. This reduction is higher than that of the single base mismatches studied (-4.9 to -7.9 kcal/mol) and is close to that observed with an adjacent double mismatch-containing duplex (-11.3 kcal/mol). The overall B-conformation of the duplex with a p-BQ-C is, however, only slightly altered, relative to the parent duplex, as shown by CD spectra. The p-BQ-C-containing duplex has been found recently to be a good substrate for the major human AP endonuclease as compared to an abasic site-containing duplex [Hang, B., et al. (1997) Biochemistry 36, 15411-15418]. We now find that the thermodynamic properties and the localized conformational changes of a p-BQ-C-containing duplex are apparently related to those reported for a duplex containing an abasic site.

Inhibition of IgE production by epsilon (epsilon) chain-specific antisense oligonucleotides (AOs) studied on human myeloma cell line U266 and peripheral blood mononuclear cells of a patient with hypereosinophilia.

Based on cDNA sequence data epsilon chain-specific antisense oligonucleotides were synthesized and checked on in vitro IgE production. Using peripheral blood cells from a hypereosinophilic patient and a human IgE myeloma cell line, U266, marked reduction of in vitro IgE production measured by PRIST was observed. The effect of epsilon antisenses proved to be isotype specific since IgG production by both peripheral blood cells and a lymphoma cell line, CESS, was not affected. Moreover, the expression of other markers on U266 (interleukin-6 receptor and gp130) were not influenced by epsilon-specific antisense oligonucleotides.

Sequence context is an important determinant in the mutagenic potential of 1,N6-ethenodeoxyadenosine (epsilonA): formation of epsilonA basepairs and elongation in defined templates.

Many laboratories have obtained data on mutagenicity of modified bases in naturally occurring DNA sequences. It has often been noted that mutation is favored in certain sequence contexts, sometimes termed 'hot spots'. This approach to the contribution of neighboring sequences does not permit a systematic study of both the qualitative and quantitative mutational frequencies. In the present experiments we have chosen to use the exocyclic adduct, 1,N6-etheno A (epsilonA), site-specifically placed in a defined 25-mer oligonucleotides in which epsilonA is flanked by differing 5' and 3' tandem bases. Mutation was assessed using an in vitro replication assay and five polymerases of varying fidelity. The relevant central sequences were 3' --> 5' -CC-epsilonA-CC-, -GG-epsilonA-GG-, -TT-epsilonA-TT-, -AA-epsilonA-AA-, -GG-epsilonA-TT-, -TT-epsilonA-AA-, -AT-epsilonA-TT- and -TA-epsilonA-TA-. Using the Klenow fragment (Kf) (exo+ or exo-) of E. coli Pol I, it was found the epsilonA is an ambiguous base and, with varying efficiencies, all four dNTPs could be inserted opposite epsilonA in all sequences. However, only 3' --> 5' -TT-epsilonA-TT-, -GG-epsilonA-TT- and -AT-epsilonA-TT- were fully extended to a significant extent. The only sequences essentially blocked at the position of epsilonA were -AA-epsilonA-AA- and -TT-epsilonA-AA-. The others were intermediate. When replication was performed with Sequenase, MMLV RT or HIV RT, different patterns were observed, in which replication terminated one base prior to epsilonA, at epsilonA, or one base after epsilonA without further extension. In favored sequences, using the Klenow fragment, an epsilonA x N pair could be extended to form normal basepairs. No extension could be demonstrated in sequences in which tandem adenines were 5' to epsilonA. Kinetic data showed that two of the epsilonA x N pairs, epsilonA x A and epsilonA x C, could form at 10 microM or less dNTP. Which bases were preferentially inserted opposite epsilonA was a function of the flanking bases. Under the kinetic conditions used, epsilonA x T did not form even at 1 mM dTTP. These results indicate that the chemical structure of an adduct is not the only determinant of mutagenic efficiency. It is likely that the effect of the adduct on replication is due to the changes in the structural environment conferred by the flanking bases.

Evidence for a common active site for cleavage of an AP site and the benzene-derived exocyclic adduct, 3,N4-benzetheno-dC, in the major human AP endonuclease.

We have previously reported that the 3,N4-benzetheno-dC (p-BQ-dC) endonuclease activity found in HeLa cells is a novel function of the major human AP endonuclease (HAP1) [Hang et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 13737-13741]. In this study, we compare the enzymatic and biochemical properties of the enzyme toward p-BQ-dC and an AP site in a defined oligonucleotide. A comparative analysis of the specificity constants (Kcat./Km) for p-BQ-dC and an AP site indicates that the AP site is the preferred substrate. The enzyme does not cleave other structurally related exocyclic adducts and modified nucleosides such as 1,N6-etheno-dA, 3,N4-etheno-dC, 1, N2-etheno-dG, 1,N2-propano-dG, 8-oxo-dG, and thymine glycol. The p-BQ-dC activity requires a double-stranded DNA substrate and is affected by the base in the opposite strand, with maximal activity for a p-BQ-dC.G pair and minimal activity for a p-BQ-dC.C pair. The p-BQ-dC activity also requires Mg2+, Mn2+, or Zn2+ with optimal concentration spectra similar to those for the AP function. The optimal pH ranges for these two functions are also similar to each other (5.5-6.5). Six mutant HAP1 proteins containing single amino acid substitutions were assayed in parallel for comparison of their activities toward p-BQ-dC and the AP site. These mutants either concomitantly lost (N212A, D210N) or had reduced (D219A, E96A, and N212Q) or unchanged (H116N) p-BQ-dC and AP activities. This parallelism strongly supports the hypothesis that cleavage of p-BQ-dC requires the same catalytic active site as that proposed for the AP function. This dual activity toward two structurally unrelated substrates, an AP site and a bulky exocyclic adduct, has implications for substrate recognition. The AP site and p-BQ-dC cause different changes in the local conformation around the lesion as it is visualized by molecular modeling.

Transvaginal ultrasonographic measurements of endometrial thickness: a reproducibility study.

OBJECTIVE: The study was undertaken to assess the reproducibility of endometrial thickness measurements by transvaginal ultrasonography (TVS). METHODS: In a prospective blind study, two examiners measured the endometrial thickness of 25 patients by TVS on two separate occasions 30 minutes apart. RESULTS: The reliability test performed for each examiner was statistically less significant for the intra-observer variation of each observer (r = 0.95 and r = 0.93), than between both examiners (r = 0.85). Although there was no statistically significant difference between the observations, the mean range of observations was 2.12 + 1.27 mm. CONCLUSIONS: A safety margin of error should be taken into consideration while recommending a cutoff under which no curettage is needed.

The unusual X-form DNA in oligodeoxynucleotides: dependence of stability on the base sequence and length.

X-form is an unusual double helix of DNA adopted by poly(dA-dT) or (dT-dA)4 at high concentrations of CsF. On the other hand, poly(dA), poly(dT), (dA-dT)4 and most other DNAs do not adopt this conformer. Here we demonstrate that the X-form is strongly destabilized by GC pairs or even minute perturbations of the alternating pyrimidinepurine sequence. For example, the 30-mer d(TATAAT)5, containing five tandem repeats of the Pribnow box, fails to isomerize into the X-form. After (dT-dA)4, the 16-mer (dT-dA)8 is shown to be the second most predisposed oligodeoxynucleotide in the (dT-dA)n series to isomerize into the X-form while the duplex lengths corresponding to n = 3,5,6,7,9,12 and 20 make the X-form unstable even in the strictly alternating (dT-dA)n sequence. Consequently, the (dT-dA)n duplex length is also a crucial factor of the X-form stability on the oligodeoxynucleotide level. We discuss a possibility that the X-form is a solution counterpart of the D-form adopted in dehydrated poly(dA-dT) fibers because properties of these two conformers are remarkably similar in many respects.

Unusual contribution of 2-aminoadenine to the thermostability of DNA.

The poly(dA-dU) and poly(dI-dC) duplexes have very similar thermostabilities (Tm). This similarity extends also to the pyrimidine 5-methyl group-containing poly(dA-dT) and poly(dI-m5dC). The differences between chemical structures of the A:U and I:C or the A:T and I:m5C base-pairs seem to be unimportant for the thermostability of the DNA. However, on the insertion of an amino group into position 2 of the purines the similarities disappear. Thermostabilities of poly(n2dA-dU) and poly(dG-dC) as well as the poly(n2dA-dT) and poly(dG-m5dC) are radically different. This is also the case with their other 5-substituted pyrimidine-containing derivatives, the 5-ethyl, 5-n-butyl and 5-bromo analogues. The G:C-based polynucleotides are more stable by an average of 40 degrees C than the n2A.U-based ones. Poly(dA,n2dA-dT)-s containing various proportions of A and n2A as well as the natural DNA of S-2L cyanophage that contains n2A bases instead of A were also studied. It was found that dependence of Tm on the n2A-content was non-linear and that the lower Tm is not the consequence of a particular nucleotide sequence. The possible structural reasons for the lower thermostabilization of these B-DNAs by the n2A:T base-pair as compared to the G:C are discussed.