Stability and structure of three-way DNA junctions containing unpaired nucleotides.

Non-paired nucleotides stabilize the formation of three-way helical DNA junctions. Two or more unpaired nucleotides located in the junction region enable oligomers ten to fifteen nucleotides long to assemble, forming conformationally homogeneous junctions, as judged by native gel electrophoresis. The unpaired bases can be present on the same strand or on two different strands. Up to five extra bases on one strand have been tested and found to produce stable junctions. The formation of stable structures is favored by the presence of a divalent cation such as magnesium and by high monovalent salt concentration. The order-disorder transition of representative three-way junctions was monitored optically in the ultraviolet and analyzed to quantify thermodynamically the stabilization provided by unpaired bases in the junction region. We report the first measurements of the thermodynamics of adding an unpaired nucleotide to a nucleic acid three-way junction. We find that delta delta G degrees (37 degrees C) = +0.5 kcal/mol for increasing the number of unpaired adenosines from two to three. Three-way junctions having reporter arms 40 base-pairs long were also prepared. Each of the three reporter arms contained a unique restriction site 15 base-pairs from the junction. Asymmetric complexes produced by selectively cleaving each arm were analyzed on native gels. Cleavage of the double helical arm opposite the strand having the two extra adenosines resulted in a complex that migrated more slowly than complexes produced by cleavage at either of the other two arms. It is likely that the strand containing the unpaired adenosines is kinked at an acute angle, forming a Y-shaped, rather than a T-shaped junction.     

Hybrid-hybrid matrix structural refinement of a DNA three-way junction from 3D NOESY-NOESY

Homonuclear 3D NOESY-NOESY has shown great promise for the structural refinement of large biomolecules. A computationally efficient hybrid-hybrid relaxation matrix refinement methodology, using 3D NOESY-NOESY data, was used to refine the structure of a DNA three-way junction having two unpaired bases at the branch point of the junction. The NMR data and the relaxation matrix refinement confirm that the DNA three-way junction exists in a folded conformation with two of the helical stems stacked upon each other. The third unstacked stem extends away from the junction, forming an acute angle (60° ) with the stacked stems. The two unpaired bases are stacked upon each other and are exposed to the solvent. Helical parameters for the bases in all three strands show slight deviations from typical values expected for right-handed B-form DNA. Inter-nucleotide imino-imino NOEs between the bases at the branch point of the junction show that the junction region is well defined. The helical stems show mobility (± 20° ) indicating dynamic processes around the junction region. The unstacked helical stem adjacent to the unpaired bases shows greater mobility compared to the other two stems. The results from this study indicate that the 3D hybrid-hybrid matrix MORASS refinement methodology, by combining the spectral dispersion of 3D NOESY-NOESY and the computational efficiency of 2D refinement programs, provides an accurate and robust means for structure determination of large biomolecules. Our results also indicate that the 3D MORASS method gives higher quality structures compared to the 2D complete relaxation matrix refinement method.     

Relative stabilities of DNA three-way, four-way and five-way junctions (multi-helix junction loops): unpaired nucleotides can be stabilizing or destabilizing.

Competition binding and UV melting studies of a DNA model system consisting of three, four or five mutually complementary oligonucleotides demonstrate that unpaired bases at the branch point stabilize three- and five-way junction loops but destabilize four-way junctions. The inclusion of unpaired nucleotides permits the assembly of five-way DNA junction complexes (5WJ) having as few as seven basepairs per arm from five mutually complementary oligonucleotides. Previous work showed that 5WJ, having eight basepairs per arm but lacking unpaired bases, could not be assembled [Wang, Y.L., Mueller, J.E., Kemper, B. and Seeman, N.C. (1991) Biochemistry, 30, 5667-5674]. Competition binding experiments demonstrate that four-way junctions (4WJ) are more stable than three-way junctions (3WJ), when no unpaired bases are included at the branch point, but less stable when unpaired bases are present at the junction. 5WJ complexes are in all cases less stable than 4WJ or 3WJ complexes. UV melting curves confirm the relative stabilities of these junctions. These results provide qualitative guidelines for improving the way in which multi-helix junction loops are handled in secondary structure prediction programs, especially for single-stranded nucleic acids having primary sequences that can form alternative structures comprising different types of junctions.     

Long-distance radical cation reactions in DNA three-way junctions: inter-arm interaction and migration through the junction

DNA three-way junctions (TWJ) are branched molecules having three ‘arms’. We studied long-distance radical cation migration in these assemblies by incorporating anthraquinone (AQ) groups linked by a covalent tether to one strand of one arm of the TWJ. Excitation of the AQ at 350 nm results in one-electron oxidation of the DNA, which generates a base radical cation. This leads to relatively inefficient (compared with duplex DNA) strand cleavage at guanines following piperidine treatment of the irradiated samples. When the AQ is linked to the 5′-terminus of arm III by a flexible tether, gel electrophoretic analysis shows that strand cleavage occurs at the guanines in all three arms. We also investigated a TWJ in which the anthraquinone is specifically intercalated in arm III. In this case, a different pattern of strand cleavage is detected. We conclude that there are at least two mechanisms for long-distance radical cation migration in TWJs: (i) by inefficient charge hopping through the junction; (ii) by a through-space, cross-arm interaction when the AQ is on a flexible tether.     

Refinement of the solution structure of a branched DNA three-way junction.

We have refined the structure of the DNA Three-Way Junction complex, TWJ-TC, described in the companion paper by quantitative analysis of two 2D NOESY spectra (mixing times 60 and 200 ms) obtained in D2O solution. NOESY crosspeak intensities extracted from these spectra were used in two kinds of refinement procedure: 1) distance-restrained energy minimization (EM) and molecular dynamics (MD) and 2) full relaxation matrix back calculation refinement. The global geometry of the refined model is very similar to that of a published, preliminary model (Leontis, 1993). Two of the helical arms of the junction are stacked. These are Helix 1, defined by basepairs S1-G1/S3-C12 through S1-C5/S3-G8 and Helix 2, which comprises basepairs S1-C6/S2-G5 through S1-G10/S2-G1. The third helical arm (Helix 3), comprised of basepairs S2-C6/S3-G5 through S2-C10/S3-G1 extends almost perpendicularly from the axis defined by Helices 1 and 2. The bases S1-C5 and S1-C6 of Strand 1 are continuously stacked across the junction region. The conformation of this strand is close to that of B-form DNA along its entire length, including the S1-C5 to S1-C6 dinucleotide step at the junction. The two unpaired bases S3-T6 and S3-C7 lie outside of the junction along the minor groove of Helix 1 and largely exposed to solvent. Analysis of the refined structure reveals that the glycosidic bond of S3-T6 exists in the syn conformation, allowing the methyl group of this residue to contact the hydrophobic surface of the minor groove of Helix 1, at S3-G11.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structures of bulged three-way DNA junctions.

We have studied a series of three-way DNA junctions containing unpaired bases on one strand at the branch-point of the junctions. The global conformation of the arms of the junctions has been analysed by means of polyacrylamide gel electrophoresis, as a function of conditions. We find that in the absence of added metal ions, all the results for all the junctions can be accounted for by extended structures, with the largest angle being that between the arms defined by the strand containing the extra bases. Upon addition of magnesium (II) or hexamine cobalt (III) ions, the electrophoretic patterns change markedly, indicative of ion-dependent folding transitions for some of the junctions. For the junction lacking the unpaired bases, the three inter-arm angles appear to be quite similar, suggesting an extended structure. However, the addition of unpaired bases permits the three-way junction to adopt a significantly different structure, in which one angle becomes smaller than the other two. These species also exhibit marked protection against osmium addition to thymine bases at the point of strand exchange. These results are consistent with a model in which two of the helical arms undergo coaxial stacking in the presence of magnesium ions, with the third arm defining an angle that depends upon the number of unpaired bases.     

NMR analysis of DNA junctions: imino proton NMR studies of individual arms and intact junction

The NMR resonances from the hydrogen-bonded guanine and thymine imino protons of base pairs in the four separate complexes forming the arms of a stable DNA four-arm junction have been assigned by using sequential nuclear Overhauser effects connecting protons in adjacent pairs. Comparison of the spectra of these individual duplex arms with that of the intact four-stranded junction suggests that base pairing occurs at the site of branching. The presence of new resonances in the spectrum of the junction can be inferred from comparison of the junction spectrum with the simulated spectra of the four individual arms. In addition, upfield shifts of the ring protons in the base pairs at the penultimate positions in the complex are observed, consistent with a change in the structure at the site of branching. These studies represent the first stage of a detailed analysis of the structure and dynamics of a DNA junction.     

Conjugation of a 3-(1H-phenanthro[9,10-d]imidazol-2-yl)-1H-indole intercalator to a triplex oligonucleotide and to a three-way junction

A new intercalating nucleic acid monomer M comprising a 4-(1-indole)-butane-1,2-diol moiety was synthesized via a classical alkylation reaction of indole-3-carboxaldehyde followed by a condensation reaction with phenanthrene-9,10-dione in the presence of ammonium acetate to form a phenanthroimidazole moiety linked to the indole ring. Insertion of the new intercalator as a bulge into a Triplex Forming Oligonucleotide resulted in good thermal stability of the corresponding Hoogsteen-type triplexes. Molecular modeling supports the possible intercalating ability of M. Hybridisation properties of DNA/DNA and RNA/DNA three-way junctions (TWJ) with M in the branching point were also evaluated by their thermal stability at pH 7. DNA/DNA TWJ showed increase in thermal stability compared to wild type oligonucleotides whereas this was not the case for RNA/DNA TWJ.     

Thermodynamic properties of an intramolecular DNA four-way junction

We have investigated the thermodynamic properties of two homologous DNA four-way junctions, J4 and J4M, based on 46-mer linear DNA molecules. J4 and J4M have the same base sequence with the only difference that the latter contains an uncharged methylene-acetal linkage, -O3'-CH2-O5', instead of the phosphodiester linkage, -O3'-PO2-O5'-, between the residues T18 and C19. The comparison of the thermal unfolding of the J4 junction and J4M junction serves to investigate the effect of the uncharged methylene-acetal linkage on the stability of the junction. Our analysis is based on CD, UV absorbance spectroscopy, DSC, and chemical footprinting. The aim is to characterize in detail the structure and stability of the junctions. As demonstrated before by NMR, in the presence of 5 mM MgCl2 +/- 50 mM NaCl, both J4 and J4M form a complete four-way junction. This is now evidenced by protection from OsO4 cleavage (chemical footprinting). We can assume that full base pairing occurs throughout the arms even at the center of the junction. CD spectra suggest that the helices within the junctions adopt the regular B-DNA conformation. Almost identical melting temperatures and unfolding enthalpies are obtained for J4 and J4M both by UV and DSC. Furthermore, the Van't Hoff enthalpy (DeltaHVH) derived from UV melting equals the calorimetric enthalpy (DeltaHcal), which means that the melting process of the structures proceeds in a two-state manner. All results taken together support the conclusion that there are no major conformational and energetic differences between J4 and J4M. The inclusion of the uncharged methylene-acetal group into the junction has no effect on its stability.     

H NMR study of a complex between the lac repressor headpiece and a 22 base pair symmetric lac operator

A complex between the lac repressor headpiece and a fully symmetric tight-binding 22 bp lac operator was studied by 2D NMR. Several 2D NOE spectra were recorded for the complex in both H2O and 2H2O. Many NOE cross-peaks between the headpiece and DNA could be identified, and changes in the chemical shift of the DNA protons upon complex formation were analyzed. Comparison of these data with those obtained for a complex between the headpiece and a 14 bp half-operator, studied previously [Boelens, R., Scheek, R. M., Lamerichs, R. M. J. N., de Vlieg, J., van Boom, J. H., & Kaptein, R. (1987) in DNA-ligand interactions (Guschlbauer, W., & Saenger, W., Eds.) pp 191-215, Plenum, New York], shows that two headpieces form a specific complex with the 22 bp lac operator in which each headpiece binds in the same way as found for the 14 bp complex. The orientation of the recognition helix in the major groove of DNA in these complexes is opposite with respect to the dyad axis to that found for other repressors.     

Solution structure of a DNA three-way junction containing two unpaired thymidine bases. Identification of sequence features that decide conformer selection

The solution structure of a DNA three-way junction (3H) containing two unpaired thymidine bases at the branch site (3HT2), was determined by NMR. Arms A and B of the 3HT2 form a quasi-continuous stacked helix, which is underwound at the junction and has an increased helical rise. The unstacked arm C forms an acute angle of approximately 55 degrees with the unique arm A. The stacking of the unpaired thymidine bases on arm C resembles the folding of hairpin loops. From this data, combined with the reported stacking behavior of 23 other 3HS2 s, two rules are derived that together correctly reproduce their stacking preference. These rules predict, from the sequence of any 3HS2, its stacking preference. The structure also suggests a plausible mechanism for structure-specific recognition of branched nucleic acids by proteins.     

Mismatch-induced DNA unbending upon duplex opening

A DNA duplex can be torn open at a specific position by introducing a branch or bulge to create an asymmetric three-way junction (TWJ). The opened duplex manifests a bent conformation (bending angle approximately 60 degrees , relative to the unopened form), which leads to a dramatic decrease in gel electrophoretic mobility. In the presence of a basepair mismatch at the opening position, the DNA backbone becomes less bent and assumes a distorted T-shaped structure, resulting in an increase in polyacrylamide gel electrophoretic mobility. Both conformational changes are confirmed using fluorescence resonance energy transfer experiments and found to be similar to the signature conformational changes of DNA duplex upon MutS protein binding. Our results imply that some structural rearrangements essential for mismatch recognition are achievable without protein interference. The gel electrophoretic mobility data for DNA TWJs with and without base mismatches correlates well with rotational diffusivity, computed by taking into account the conformational change of TWJ induced by base mismatch.     

Structure of Three-Way DNA Junctions 1. Non-Planar DNA Geometry

Abstract

Three-way junctions were obtained by annealing two synthetic DNA-oligomers. One of the strands contains a short palindrome sequence, leading to the formation of a hairpin with four base pairs in the stem and four bases in the loop. Another strand is complementary to the linear arms of the first hairpin-containing strand. Both strands were annealed to form a three-way branched structure with sticky ends on the linear arms. The branched molecules were ligated, and the ligation mixture was analysed on a two-dimensional gel in conditions which separated linear and circular molecules. Analysis of 2D-electrophoresis data shows that circular molecules with high mobility are formed. Formation of circular molecules is indicative of bends between linear arms. We estimate the magnitude of the angle between linear arms from the predominant size of the circular molecules formed. When the junction-to-junction distance is 20–21 bp, trimers and tetramers are formed predominately, giving an angle between linear arms as small as 60–90°. Rotation of the hairpin position in the three- way junction allowed us to measure angles between other arms, yielding similar values. These results led us to conclude that the three-way DNA junction possesses a non-planar pyramidal geometry with 60–90° between the arms. Computer modeling of the three-way junction with 60° pyramidal geometry showed a predominantly B-form structure with local distortions at the junction points that diminish towards the ends of the helices. The size distributions of circular molecules are rather broad indicating a dynamic flexibility of three-way DNA junctions.     

Influence of minor groove substituents on the structure of DNA Holliday junctions

The inosine-containing sequence d(CCIGTACm(5)CGG) is shown to crystallize as a four-stranded DNA junction. This structure is nearly identical to the antiparallel junction formed by the parent d(CCGGTACm(5)()CGG) sequence [Vargason, J. M., and Ho, P. S. (2002) J. Biol. Chem. 277, 21041-21049] in terms of its conformational geometry, and inter- and intramolecular interactions within the DNA and between the DNA and solvent, even though the 2-amino group in the minor groove of the important G(3).m(5)C(8) base pair of the junction core trinucleotide (italicized) has been removed. In contrast, the analogous 2,6-diaminopurine sequence d(CCDGTACTGG) crystallizes as resolved duplex DNAs, just like its parent sequence d(CCAGTACTGG) [Hays, F. A., Vargason, J. M., and Ho, P. S. (2003) Biochemistry 42, 9586-9597]. These results demonstrate that it is not the presence or absence of the 2-amino group in the minor groove of the R(3).Y(8) base pair that specifies whether a sequence forms a junction, but the positions of the extracyclic amino and keto groups in the major groove. Finally, the study shows that the arms of the junction can accommodate perturbations to the B-DNA conformation of the stacked duplex arms associated with the loss of the 2-amino substituent, and that two hydrogen bonding interactions from the C(7) and Y(8) pyrimidine nucleotides to phosphate oxygens of the junction crossover specify the geometry of the Holliday junction.     

Global structure of a DNA three-way junction by solution NMR: towards prediction of 3H fold

Three-way junctions (3H) are the simplest and most commonly occurring branched nucleic acids. They consist of three double helical arms (A to C), connected at the junction point, with or without a number of unpaired bases in one or more of the three different strands. Three-way junctions with two unpaired bases in one strand (3HS2) have a high tendency to adopt either of two alternative stacked conformations in which two of the three arms A, B and C are coaxially stacked, i.e. A/B-stacked or A/C-stacked. Empirical stacking rules, which successfully predict for DNA 3HS2 A/B-stacking preference from sequence, have been extended to A/C-stacked conformations. Three novel DNA 3HS2 sequences were designed to test the validity of these extended stacking rules and their conformational behavior was studied by solution NMR. All three show the predicted A/C-stacking preference even in the absence of multivalent cations. The stacking preference for both classes of DNA 3HS2 can thus be predicted from sequence. The high-resolution NMR solution structure for one of the stacked 3HS2 is also reported. It shows a well-defined local and global structure defined by an extensive set of classical NMR restraints and residual dipolar couplings. Analysis of its global conformation and that of other representatives of the 3H family, shows that the relative orientations of the stacked and non-stacked arms, are restricted to narrow regions of conformational space, which can be understood from geometric considerations. Together, these findings open up the possibility of full prediction of 3HS2 conformation (stacking and global fold) directly from sequence.     

NMR structural studies of intramolecular (Y+)n.(R+)n(Y-)nDNA triplexes in solution: imino and amino proton and nitrogen markers of G.TA base triple formation.

We reported previously on NMR studies of (Y+)n.(R+)n(Y-)n DNA triple helices containing one oligopurine strand (R)n and two oligopyrimidine strands (Y)n stabilized by T.AT and C+.GC base triples [de los Santos, C., Rosen, M., & Patel, D. J. (1989) Biochemistry 28, 7282-7289]. Recently, it has been established that guanosine can recognize a thymidine.adenosine base pair to form a G.TA triple in an otherwise (Y+)n.(R+)n(Y-)n triple-helix motif. [Griffin, L. C., & Dervan, P. B. (1989) Science 245, 967-971]. The present study extends the NMR research to the characterization of structural features of a 31-mer deoxyoligonucleotide that folds intramolecularly into a 7-mer (Y+)n.(R+)n(Y-)n triplex with the strands linked through two T5 loops and that contains a central G.TA triple flanked by T.AT triples. The G.TA triplex exhibits an unusually well resolved and narrow imino and amino exchangeable proton and nonexchangeable proton spectrum in H2O solution, pH 4.85, at 5 degrees C. We have assigned the imino protons of thymidine and amino protons of adenosine involved in Watson-Crick and Hoogsteen pairing in T.AT triples, as well as the guanosine imino and cytidine amino protons involved in Watson-Crick pairing and the protonated cytidine imino and amino protons involved in Hoogsteen pairing in C+.GC triples in the NOESY spectrum of the G.TA triplex. The NMR data are consistent with the proposed pairing alignment for the G.TA triple where the guanosine in an anti orientation pairs through a single hydrogen bond from one of its 2-amino protons to the 4-carbonyl group of thymidine in the Watson-Crick TA pair.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA hairpin structures in solution: 500-MHz two-dimensional 1H NMR studies on d(CGCCGCAGC) and d(CGCCGTAGC)

A hairpin structure contains two conformationally distinct domains: a double-helical stem with Watson-Crick base pairs and a single-stranded loop that connects the two arms of the stem. By extensive 1D and 2D 500-MHz 1H NMR studies in H2O and D2O, it has been demonstrated that the DNA oligomers d(CGCCGCAGC) and d(CGCCGTAGC) form hairpin structures under conditions of low concentration, 0.5 mM in DNA strand, and low salt (20 mM NaCl, pH 7). From examination of the nuclear Overhauser effect (NOE) between base protons H8/H6 and sugar protons H1' and H2'/H2", it was concluded that in d(CGCCGCAGC) and d(CGCCGTAGC) all the nine nucleotides display average (C2'-endo,anti) geometry. The NMR data in conjunction with molecular model building and solvent accessibility studies were used to derive a working model for the hairpins.     

Comparative NMR study of A(n)-bulge loops in DNA duplexes: intrahelical stacking of A, A-A, and A-A-A bulge loops.

We have prepared a series of deoxyoligonucleotide duplexes of the sequence d(G-C-A-T-C-G-X-G-C-T-A-C-G).d(C-G-T-A-G-C-C-G-A-T-G-C), in which X represents either one (A), two (A-A), or three (A-A-A) unpaired adenine basis. Using two-dimensional proton and phosphorus NMR spectroscopy, we have characterized conformational features of these bulge-loop duplexes in solution. We find that Watson-Crick hydrogen bonding is intact for all 12 base pairs, including the GC bases that flank the bulge loop. Observation of NOE connectivities in both H2O and D2O allows us to unambiguously localize all of the bulged adenine residues to intrahelical positions within the duplex. This is in contrast to an earlier model for multiple-base bulge loops in DNA [Bhattacharyya, A., & Lilley, D. M. J. (1989) Nucleic Acids Res. 17, 6821-6840], in which all but the most 5' bulged base are looped out into solution. We find that insertion of two or three bases into the duplex results in the disruption of specific sequential NOEs for the base step across from the bulge loop site on the opposite strand. This disruption is characterized by a partial shearing apart of these bases, such that certain sequential NOEs for this base step are preserved. We observe a downfield-shifted phosphorus resonance, which we assign in the A-A-A bulge duplex to the 3' side of the last bulged adenine residue. Proton and phosphorus chemical shift trends within the An-bulge duplex series indicate that there is an additive effect on the structural perturbations caused by additional unpaired bases within the bulge loop. This finding parallels previous observations [Bhattacharyya, A., & Lilley, D. M. J. (1989) Nucleic Acids Res. 17, 6821-6840; Hsieh, C.-H., & Griffith, J. D. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4833-4837] on the magnitude of the induced bending of DNA duplexes by multiple-base bulge loops.     

The identification of the A-type RNA helices in a 55mer RNA by selective incorporation of deuterium-labelled nucleotide residues (Uppsala NMR-window concept)

The 55-nt long RNA, modelling a three-way junction, with non-uniformly incorporated deuterated nucleotides has been synthesised in a pure form. The NMR-window part in this partially deuterated 55mer RNA consists of natural non-enriched nucleotide blocks at the three-way junction (shown in a square box in Fig. 2), whereas all other nucleotides of the rest of the molecule are partially deuterated (> 97 atom% 2H at C2', C3', C5', C5, and approximately 50 atom% 2H at C4'). The secondary structure of this 55mer RNA was determined by 2D 1H NOESY spectroscopy in D2O or in 10% D2O-H2O mixture. The use of deuterated building blocks in the specific region of the 55mer RNA allowed us to identify two distinct A-type RNA helices in a straightforward manner by observing connectivities of H1' with the basepaired imino and the aromatic H2 of all adenosine nucleotides as the first step for the determination of its tertiary structure in a cost- and time-effective manner without employing any 13C/15N labelling. These two decameric helices involve 40 nucleotides, for which all non-exchangeable H1', H6, H2, H8 and H5 protons (all 40 H1', all 40 H6 or H8 aromatics, all seven H2 of adenine nucleotide and all four non-deuterated H5 of cytosines) as well as all 16 exchangeable imino protons (with the exception of four terminal basepairs) and 16 amino protons of cytosines have been assigned. Since all aromatic-H2', H3' as well as H5'/5' crosspeaks from partially deuterated residues have been eliminated from the NMR spectra, the observation of natural nucleotide residues in the NMR window part has essentially been simplified. It has been found that the crosspeaks from the natural nucleotides located at the three-way junction in the NMR-window part show different degrees of line-broadening, thereby indicating that the various nucleotide residues have very different mobilities with respect to themselves as well as compared to other nucleotides in the helices. The assignment of H2' and H3' in the NMR-window part has been made based on NOESY and DQF-COSY crosspeaks. It is noteworthy that, even in this preliminary study, it has been possible to identify 10 H2' out of total 14 and 9 H3' out of 14. The data show that expanded AU containing a tract of 55mer RNA does not self-organise into a tight third helix, as the two decameric A-type helices, across the three-way junction which is evident from the absence of any additional imino protons, except those that already have been assigned for the two decameric helices.     

Improved Targeting of the Flanks of a DNA Stem Using α-Oligodeoxynucleotides.-The Enhanced Effect of an Intercalator

Abstract

2′-Deoxy-5′-0-(4,4′-dimethoxytrityl)-5-methyl-N ⁴-(1-pyrenylmethyl)-α-cytidine (5) was prepared by reaction of 1-pyrenylmethylamine with an appropriate protected 4-(l,2,4-triazolyl)-α-thymidine derivative 3 which was synthesized from 5-O-DMT protected α-thymidine 1. Aminolysis of 3 afforded 3′-O-acetyl-2′-deoxy-5′-O-(4,4′-dimethoxytrityl)-5-methyl-α-cytidine (8). Benzoylation of 8 and removal of acetyl afforded N ⁴-benzoyl-2-deoxy-5–0-(4,4′-dimethoxytrityl)-5-methyl-α-cytidine (10). The amidites of compounds 5and 10 were prepared and used in α-oligonucleotide synthesis. DNA three-way junction (TWJ) is stabilized when an α-ODN is used for targeting the dangling flanks of the stem in a DNA hairpin. Further stabilization of the TWJ is observed when 5 is inserted into the α-ODN at the junction region.