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.     

Properties of triple helix formation with oligodeoxyribonucleotides containing 8-oxo-2′-deoxyadenosine and 2′-modified nucleoside derivatives

The ability of homopyrimidine oligonucleotides containing 8-oxo-2′-deoxyadenosine (dAOH), 2′-methoxyuridine (Um). 2′-fluorouridine (Uf), 2′-methoxycytidine (Cm), and 2′-fluorocytidine (Cf) to form stable, triple-helical structures with sequences containing the recognition site for the class II-S restriction enzyme, Ksp632-I, was studied as a function of pH. The 8-oxo-2′-deoxyadenosine substituted oligomers were shown to bind within the physiological pH range in a pH-independent fashion, without a compromise in specificity. In particular, the substitutions of three deoxycytidine residues with 8-oxo-2′-deoxyadenosine showed higher endonuclease inhibition than the substitution of either one or two deoxycytidine residues with 8-oxo-2′-deoxyadenosine. In contrast, the oligonucleotides containing 2′-modified nucleosides (Uf, Um, Uf-Cf, Um-Cm, dAOHUf, and dAOH-Um) bind in a pH-dependent manner to the target duplex.

The 8-oxo-2′-deoxyadenosine substituted oligomers were shown to bind within the physiological pH range in a pH-independent fashion, without a compromise in specificity. In particular, the substitutions of three deoxycytidine resides with 8-oxo-2′-deoxyadenosine showed higher endonuclease inhibition than the substitution of either one or two deoxycytidine residues with 8-oxo-2′-deoxyadenosine. By contrast, the oligonucleotides containing 2′-modified nucleosides (Uf, Um, Uf-Cf, Um-Cm, dAOH-Uf, and dAOH-Um) bind in a pH-dependent manner to the target duplex.     

Synthesis of a dA-dT base pair analogue and its effects on DNA-ligand binding

Two nucleoside derivatives containing the base analogues 3-deazaadenine and 3-methyl-2-pyridone have been prepared as analogues of dA and dT, respectively. After conversion into the appropriately protected phosphoramidites, DNA sequences were prepared with site-specifically placed analogues. When present in a duplex DNA sequence, the analogues result in the deletion of one or both of the hydrogen bonding functional groups (the N3-nitrogen of dA and the O2-carbonyl of dT) present in the minor groove. Binding by two ligands, 4',6-diamidine-2-phenyl indole (DAPI) and Hoechst 33258 in the minor groove has been probed using a variety of DNA sequences. These sequences contain a d(GAATTC)2 core with analogue nucleosides substituted for one or more of the dA and dT residues. DAPI bound strongly to any sequence that contained both O2-carbonyls of the central two dT residues. The presence of a dc3A residue did in some cases enhance binding. With one of the central O2-carbonyls deleted, the binding was noticeably reduced, and with both absent, no significant binding could be detected. Similar although less dramatic results were observed with Hoechst 33258 binding to analogue sequences.     

The mechanism of DNA repair by uracil-DNA glycosylase: studies using nucleotide analogues

2',4'-Dideoxy-4'-methyleneuridine incorporated into oligodeoxynucleotides forms regular B-DNA duplexes as shown by Tm and CD measurements. Such oligomers are not cleaved by the DNA repair enzyme, UDG, which cleaves the glycosylic bond in dU but not in dT nor in dC nucleosides in single stranded and double stranded DNA. Differential binding of oligomers containing carbadU, 4'-thiodU, and dU residues to wild type and mutant UDG proteins identify an essential role for the furanose 4'-oxygen in recognition and cleavage of dU residues in DNA.     

7-Deaza-2'-deoxyadenosine and 3-deaza-2'-deoxyadenosine replacing dA within d(A6)-tracts: differential bending at 3'- and 5'-junctions of d(A6).d(T6) and B-DNA.

7-Deaza-2'-deoxyadenosine (1, c7Ad) and 3-deaza-2'-deoxyadenosine (2, c3Ad) have been incorporated into d(AAAAAA) tracts replacing dA at various positions within oligonucleotides. For this purpose suitably protected phosphonates have been prepared and oligonucleotides were synthesized on solid-phase. The oligomers were hybridized with their cognate strands. The duplexes were phosphorylated at OH-5' by polynucleotide kinase and self-ligated to multimers employing T4 DNA ligase. Oligomerized DNA-fragments were analyzed by polyacrylamide gel electrophoresis and the bending was determined from anomalies of electrophoretic mobility. Replacement of dA by c3Ad decreased the bending more than replacement by c7Ad. Reduction of bending was much stronger when the modified nucleosides replaced one or several dA residues at the 3'-site of an d(AAAAAA)-tract whereas replacement at the 5'-site showed no significant influence [1, 2].     

Properties of 2'-fluorothymidine-containing oligonucleotides: interaction with restriction endonuclease EcoRV

2'-Fluorothymidine (Tf) was synthesized via an improved procedure with (diethylamino)sulfur trifluoride. The compatibility of the analogue with DNA synthesis via the phosphoramidite method was demonstrated after complete enzymatic digestion of the oligonucleotides d(Tf11T) and d(Tf3T), the sole products of which were 2'-fluorothymidine and thymidine in the expected ratio. The 2'-fluorothymidine was also incorporated into the EcoRV recognition sequence (underlined), within the complementary oligonucleotides d(CAAACCGATATCGTTGTG) and d(CACAACGATATCGGTTTG). Thermal melting characteristics of these duplexes showed a significant decrease in stability only when both of the thymidine residues in one of the strands were replaced. In contrast, when all of one strand of a duplex contained 2'-fluorothymidine, as in d(Tf11T).d(A12), a substantially higher Tm and cooperativity of melting was observed relative to the unmodified structure. EcoRV cleaved a duplex that contained a 2'-fluorothymidine at the scissile linkage in each strand at two-thirds of the rate obtained for the unmodified structure. A duplex containing two 2'-fluorothymidine residues in one strand and none in the other was cleaved at one-third of the rate in its unsubstituted strand, whereas the cleavage rate was reduced to 22% in its modified strand.     

The Mechanism of Dna Repair by Uracil-Dna Glycosylase: Studies Using Nucleotide Analogues

Abstract

2′,4′-Dideoxy-4′-methyleneuridine incorporated into oligodeoxynucleotides forms regular B-DNA duplexes as shown by T_m and CD measurements. Such oligomers are not cleaved by the DNA repair enzyme, UDG, which cleaves the glycosylic bond in dU but not in dT nor in dC nucleosides in single stranded and double stranded DNA. Differential binding of oligomers containing carbadU, 4′-thiodU, and dU residues to wild type and mutant UDG proteins identify an essential role for the furanose 4′-oxygen in recognition and cleavage of dU residues in DNA.     

Synthesis and Some Properties of Modified Oligonucleotides. II. Oligonucleotides Containing 2′-Deoxy-2′-fluoro-β-D-arabinofuranosyl Pyrimidine Nucleosides

Abstract

In order to find the effects of unnatural nucleosides on the stability of duplex, several oligonucleotides containing 1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-uracil(FAU),-cytosine (FAC) and -thymine (FMAU) were synthesized by two alternative approaches: phosphoramidite method on an ABI 392 synthesizer and H-phosphonate procedure on our GeneSyn I universal module synthesizer. It was shown from the melting profiles that the presence of FMAU has a large stabilizing effect on the duplex. Replacement of thymidine with FAU, or deoxycytidine with FAC resulted in the formation of less stable duplexes. Temperature-dependent CD spectroscopy demonstrated that the structures of the fluorine containing oligomers are very similar to those of unmodified oligomers.     

Interactions of oligonucleotide analogs containing methylphosphonate internucleotide linkages and 2'-O-methylribonucleosides.

The interactions of oligonucleotide analogs, 12-mers, which contain deoxyribo- or 2'-O-methylribose sugars and methylphosphonate internucleotide linkages with complementary 12-mer DNA and RNA targets and the effect of chirality of the methylphosphonate linkage on oligomer-target interactions was studied. Oligomers containing a single Rp or Sp methylphosphonate linkage (type 1) or oligomers containing a single phosphodiester linkage at the 5'-end followed by 10 contiguous methylphosphonate linkages of random chirality (type 2) were prepared. The deoxyribo- and 2'-O-methylribo- type 1 12-mers formed stable duplexes with both the RNA and DNA as determined by UV melting experiments. The melting temperatures, Tms, of the 2'-O-methylribo-12-mer/RNA duplexes (49-53 degrees C) were higher than those of the deoxyribo-12mer/RNA duplexes (31-36 degrees C). The Tms of the duplexes formed by the Rp isomers of these oligomers were approximately 3-5 degrees C higher than those formed by the corresponding Sp isomers. The deoxyribo type 2 12-mer formed a stable duplex, Tm 34 degrees C, with the DNA target and a much less stable duplex with the RNA target, Tm < 5 degrees C. In contrast, the 2'-O-methylribo type 2 12-mer formed a stable duplex with the RNA target, Tm 20 degrees C, and a duplex of lower stability with the DNA target, Tm < 5 degrees C. These results show that the previously observed greater stability of oligo-2'-O-methylribonucleotide/RNA duplexes versus oligodeoxyribonucleotide/RNA duplexes extends to oligomers containing methylphosphonate linkages and that the configuration of the methylphosphonate linkage strongly influences the stability of the duplexes.     

Effect of base-pair sequence on the conformations and thermally induced transitions in oligodeoxyribonucleotides containing only AT base pairs

Tm curves, CD spectra, and kinetics results of the self-complementary DNA dodecamers d(A6T6), d(A3T3A3T3), d(A2T2A2T2A2T2), d(ATATATATATAT), and d(T6A6) demonstrate that the thermal transitions of these oligomers at low salt concentration involve a hairpin intermediate. At high salt concentrations (greater than 0.1 M Na+) only a duplex to denatured-strand transition appears to occur. The temperature and salt-concentration regions of the transitions are very sequence dependent. Alternating-type AT sequences have a lower duplex stability and a greater tendency to form hairpins than sequences containing more nonalternating AT base pairs. Of the two nonalternating sequences, d(T6A6) is significantly less stable than d(A6T6). Both oligomers have CD curves that are very similar to the unusual CD spectrum of poly(dA).poly(dT). The Raman spectra of these two oligomers are also quite similar, but at low temperature, small intensity differences in two backbone modes and three nucleoside vibrations are obtained. The hairpin to duplex transition for the AT dodecamers was examined by salt-jump kinetics measurements. The transition is faster than transitions for palindromic-sequence oligomers containing terminal GC base pairs. Stopped-flow kinetics studies indicate that the transition is second order and has a relatively low activation energy. The reaction rate increases with increasing ionic strength. These results are consistent with a three-step mechanism for the hairpin to duplex reaction: (i) fraying of the hairpin oligomers' terminal base pairs, (ii) a rate-determining bimolecular step involving formation of a cruciform-type intermediate from two hairpin oligomers with open terminal base pairs, and (iii) base-pair migration and formation in the intermediate to give the duplex.     

Nucleosides and oligonucleotides containing 1,2,3-triazole residues with nucleobase tethers: synthesis via the azide-alkyne 'click' reaction

A series of novel 1,2,3-triazole nucleosides linked to DNA nucleobases were prepared via copper(I)-catalyzed 1,3-dipolar cycloaddition of N-9 propargylpurines or N-1 propargylpyrimidines with the tolouyl protected 1-azido-2-deoxyribofuranose 2 followed by treatment with NaOMe/MeOH or aq NH3. The antiviral activity of such compounds against selected RNA viruses is reported. The strongly fluorescent 1,2,3-triazole compounds 16 and 17 were synthesized from propargylated uracil 1a and propargylated adenine 1c with coumarin azide 15, and the fluorescence properties were studied. The nucleosides 4 and 6 were incorporated into DNA using the phosphoramidite building blocks and employed in solid-phase synthesis. Melting experiments demonstrated that such 1,2,3-triazole nucleosides have a negative impact on the duplex stability when they are placed opposite to the canonical bases as well as abasic sites. The nucleobases attached to the triazole ring cannot involve in the base pair formation with the opposite bases because of the structural variations induced by the triazole ring. The stacking of such nucleosides when positioned at the end of oligonucleotides retains the stability of DNA duplexes. The duplex structures were studied by molecular modelling which support the results of melting experiments.     

Zwitterionic oligodeoxyribonucleotide N3'-->P5' phosphoramidates: synthesis and properties.

Zwitterionic, net neutral oligonucleotides containing alternating negatively charged N3'-->P5' phosphoramidate monoester and positively charged phosphoramidate diester groups were synthesized. The ability of zwitterionic phosphoramidates to form complexes with complementary DNA and RNA was evaluated. Stoichiometry and salt dependency of these complexes were determined as a function of the nature of the heterocyclic bases of the zwitterionic compounds. Unlike the melting temperatures of the natural phosphodiester-containing oligomers, the T m of the duplexes formed with the zwitterionic oligothymidylates was salt concentration independent. The thermal stability of these duplexes was much higher with Delta T m values of 20-35 degrees C relatively to phosphodiester counterparts at low salt concentrations. The zwitterionic oligoadenylate formed only 2Py:1Pu triplexes with complementary poly(U) or poly(dT) strands. The thermal stability of these complexes was dependent on salt concentration. Also, the T m values of the complexes formed by the zwitterionic oligoadenylate with poly(U) were 6-41 degrees C higher than for the natural phosphodiester counterpart. Triplexes of this compound with poly(dT) were also more stable with a Delta T m value of 22 degrees C at low salt concentrations. Complexes formed by the zwitterionic oligonucleotides with complementary RNAs were not substrates for RNase H. Surprisingly, the duplex formed by the all anionic alternating N3'-->P5'phosphoramidate-phosphodiester oligothymidylate and poly(A) was a good substrate for RNase H.     

Synthesis and properties of oligonucleotides containing a 7-membered (oxepane) sugar ring

Herein we describe the synthesis of novel 7-membered ring (oxepane) thymine and adenine nucleosides (oT and oA) and their corresponding 5'-O-phosphoramidite derivatives. Two homopolymeric sequences (oT(15) and oA(15)) were prepared via conventional solid-phase synthesis. The mutually complementary strands had the ability to form a duplex (oT(15):oA(15)) exhibiting a transition temperature of 12 degrees C. The oxepane oligonucleotides were also found to associate with their respective complementary RNA strands thus forming oT(15):rA(15) (13 degrees C) and oA(15):rU(15) (12 degrees C) hybrids. The corresponding native duplexes, namely dT(15):dA(15), dT(15):rA(15) and dA(15):rU(15) had melting temperatures of 37 degrees C, 32 degrees C and 16 degrees C, respectively. The CD spectrum of oT(15):rA(15) closely resembled that of the native dT(15):rA(15) hybrid and, in fact, both were found to be substrates for E. Coli RNase H. Thus the oxepane nucleic acids reported here are one of only a handful of DNA mimics capable of activating RNase H when bound to RNA.     

A magnesium-induced conformational transition in the loop of a DNA analog of the yeast tRNA(Phe) anticodon is dependent on RNA-like modifications of the bases of the stem.

Two single-stranded DNA heptadecamers corresponding to the yeast tRNA(Phe) anticodon stem-loop were synthesized, and the solution structures of the oligonucleotides, d(CCAGACTGAAGATCTGG) and d(CCAGACTGAAGAU-m5C-UGG), were investigated using spectroscopic methods. The second, or modified, base sequence differs from that of DNA by RNA-like modifications at three positions; dT residues were replaced at positions 13 and 15 with dU, and the dC at position 14 with d(m5C), corresponding to positions where these nucleosides occur in tRNA(Phe). Both oligonucleotides form intramolecular structures at pH 7 in the absence of Mg2+ and undergo monophasic thermal denaturation transitions (Tm = 47 degrees C). However, in the presence of 10 mM Mg2+, the modified DNa adopted a structure that exhibited a biphasic "melting" transition (Tm values of 23 and 52 degrees C) whereas the unmodified DNA structure exhibited a monophasic denaturation (Tm = 52 degrees C). The low-temperature, Mg(2+)-dependent structural transition of the modified DNA was also detected using circular dichroism (CD) spectroscopy. No such transition was exhibited by the unmodified DNA. This transition, unique to the modified DNA, was dependent on divalent cations and occurred most efficiently with Mg2+; however, Ca2+ also stabilized the alternative conformation at low temperature. NMR studies showed that the predominant structure of the modified DNA in sodium phosphate (pH 7) buffer in the absence of Mg2+ was a hairpin containing a 7-nucleotide loop and a stem composed of 3 stable base pairs. In the Mg(2+)-stabilized conformation, the loop became a two-base turn due to the formation of two additional base pairs across the loop.(ABSTRACT TRUNCATED AT 250 WORDS)     

2'-modified nucleosides for site-specific labeling of oligonucleotides.

We report the synthesis of 2'-modified nucleosides designed specifically for incorporating labels into oligonucleotides. Conversion of these nucleosides to phosphoramidite and solid support-bound derivatives proceeds in good yield. Large-scale synthesis of 11-mer oligonucleotides possessing the 2'-modified nucleosides is achieved using these derivatives. Thermal denaturation studies indicate that the presence of 2'-modified nucleosides in 11-mer duplexes has minimal destabilizing effects on the duplex structure when the nucleosides are placed at the duplex termini. The powerful combination of phosphoramidite and support-bound derivatives of 2'-modified nucleosides affords the large-scale preparation of an entirely new class of oligonucleotides. The ability to synthesize oligonucleotides containing label attachment sites at 3', intervening, and 5' locations of a duplex is a significant advance in the development of oligonucleotide conjugates.     

Comparison of the RNase H cleavage kinetics and blood serum stability of the north-conformationally constrained and 2'-alkoxy modified oligonucleotides

The RNase H cleavage potential of the RNA strand basepaired with the complementary antisense oligonucleotides (AONs) containing North-East conformationally constrained 1',2'-methylene-bridged (azetidine-T and oxetane-T) nucleosides, North-constrained 2',4'-ethylene-bridged (aza-ENA-T) nucleoside, and 2'-alkoxy modified nucleosides (2'-O-Me-T and 2'-O-MOE-T modifications) have been evaluated and compared under identical conditions. When compared to the native AON, the aza-ENA-T modified AON/RNA hybrid duplexes showed an increase of melting temperature (DeltaTm = 2.5-4 degrees C per modification), depending on the positions of the modified residues. The azetidine-T modified AONs showed a drop of 4-5.5 degrees C per modification with respect to the native AON/RNA hybrid, whereas the isosequential oxetane-T modified counterpart, showed a drop of approximately 5-6 degrees C per modification. The 2'-O-Me-T and 2'-O-MOE-T modifications, on the other hand, showed an increased of Tm by 0.5 C per modification in their AON/RNA hybrids. All of the partially modified AON/RNA hybrid duplexes were found to be good substrates for the RNase H mediated cleavage. The Km and Vmax values obtained from the RNA concentration-dependent kinetics of RNase H promoted cleavage reaction for all AON/RNA duplexes with identical modification site were compared with those of the reference native AON/RNA hybrid duplex. The catalytic activities (Kcat) of RNase H were found to be greater (approximately 1.4-2.6-fold) for all modified AON/RNA hybrids compared to those for the native AON/RNA duplex. However, the RNase H binding affinity (1/Km) showed a decrease (approximately 1.7-8.3-fold) for all modified AON/RNA hybrids. This resulted in less effective (approximately 1.1-3.2-fold) enzyme activity (Kcat/Km) for all modified AON/RNA duplexes with respect to the native counterpart. A stretch of five to seven nucleotides in the RNA strand (from the site of modifications in the complementary modified AON strand) was found to be resistant to RNase H digestion (giving a footprint) in the modified AON/RNA duplex. Thus, (i) the AON modification with azetidine-T created a resistant region of five to six nucleotides, (ii) modification with 2'-O-Me-T created a resistant stretch of six nucleotides, (iii) modification with aza-ENA-T created a resistant region of five to seven nucleotide residues, whereas (iv) modification with 2'-O-MOE-T created a resistant stretch of seven nucleotide residues. This shows the variable effect of the microstructure perturbation in the modified AON/RNA heteroduplex depending upon the chemical nature as well as the site of modifications in the AON strand. On the other hand, the enhanced blood serum as well as the 3'-exonuclease stability (using snake venom phosphodiesterase, SVPDE) showed the effect of the tight conformational constraint in the AON with aza-ENA-T modifications in that the 3'-exonuclease preferentially hydrolyzed the 3'-phosphodiester bond one nucleotide away (n + 1) from the modification site (n) compared to all other modified AONs, which were 3'-exonuclease cleaved at the 3'-phosphodiester of the modification site (n). The aza-ENA-T modification in the AONs made the 5'-residual oligonucleotides (including the n + 1 nucleotide) highly resistant in the blood serum (remaining after 48 h) compared to the native AON (fully degraded in 2 h). On the other hand, the 5'-residual oligonucleotides (including the n nucleotide) in azetidine-T, 2'-O-Me-T, and 2'-O-MOE-T modified AONs were more stable compared to that of the native counterpart but more easily degradable than that of aza-ENA-T containing AONs.     

Parallel-stranded duplex DNA containing dA · dU base pairs

DNA oligonucleotides with dA and dU residues can form duplexes with trans d(A · U) base pairing and the sugar-phosphate backbone in a parallel-stranded orientation, as previously established for oligonucleotides with d(A · T) base pairs. The properties of such parallel-stranded DNA (ps-DNA) 25-mer duplexes have been characterized by absorption (uv), CD, ir, and fluorescence spectroscopy, as well as by nuclease sensitivity. Comparisons were made with duplex molecules containing (a) dT in both strands, (b) dU in one strand and dT in the second, and (c) the same base combinations in reference antiparallel-stranded (aps) structures. Thermodynamic analysis revealed that total replacement of deoxythymine by deoxyuridine was accompanied by destabilization of the ps-helix (reduction in T_m by −13°C in 2 mM MgGl₂, 10 mM Na-cacodylate). The U-containing ps-helix (U1 · U2) also melted 14°C lower than the corresponding aps-helix under the same ionic conditions; this difference was very close to that observed between ps and aps duplexes with d(A · T) base pairs. Force field minimized structures of the various ps and aps duplexes with either d(A · T) or d(A · U) base pairs ps/aps and dT/dU combinations are presented. The energy-minimized helical parameters did not differ significantly between the DNAs containing dT and dU. © 1996 John Wiley & Sons, Inc.     

Azole substituted oligonucleotides promote antiparallel triplex formation at non-homopurine duplex targets.

The ability of certain azole substituted oligodeoxy-ribonucleotides to promote antiparallel triple helix formation with duplex targets having CG or TA interruptions in the otherwise homopurine sequence was examined. 2'-Deoxyribonucleosides of the azoles, which include pyrazole, imidazole, 1,2,4-triazole and 1,2,3,4-tetrazole were synthesized using the stereo-specific sodium salt glycosylation procedure. These nucleosides were successfully incorporated using solid-support, phosphoramidite chemistry, into oligonucleotides designed to interact with the non-homopurine duplex targets. The interaction of these modified oligonucleotides with all four possible base pairs was evaluated and compared to similar data for a series of natural oligonucleotides. The oligonucleotides containing simple azoles enhanced the triplex forming ability considerably at non-homopurine targets. Binding of these modified oligonucleotides to duplex targets containing TA inversion sites was particularly noteworthy, and compare favorably to unmodified oligonucleotides for binding to duplex targets containing CG as well as TA base pairs. The selectivity exhibited by certain azoles is suggestive of base pair specific interactions. Thus, the azoles evaluated during this study show considerable promise for efforts to develop generalized triplex formation at non-homopurine duplex sequences.     

Chemically modified oligonucleotides with efficient RNase H response

Ten different chemically modified nucleosides were incorporated into short DNA strands (chimeric oligonucleotides ON3-ON12 and ON15-ON24) and then tested for their capacity to mediate RNAse H cleavage of the complementary RNA strand. The modifications were placed at two central positions directly in the RNase H cleaving region. The RNA strand of duplexes with ON3, ON5 and ON12 were cleaved more efficiently than the RNA strand of the DNA:RNA control duplex. There seems to be no correlation between the thermal stability between the duplexes and RNase H cleavage.     

A parallel stranded linear DNA duplex incorporating dG.dC base pairs

DNA oligonucleotides with appropriately designed complementary sequences can form a duplex in which the two strands are paired in a parallel orientation and not in the conventional antiparallel double helix of B-DNA. All parallel stranded (ps) molecules reported to date have consisted exclusively of dA.dT base pairs. We have substituted four dA.dT base pairs of a 25-nt parallel stranded linear duplex (ps-D1.D2) with dG.dC base pairs. The two strands still adopt a duplex structure with the characteristic spectroscopic properties of the ps conformation but with a reduced thermodynamic stability. Thus, the melting temperature of the ps duplex with four dG.dC base pairs (ps-D5.D6) is 10-16 degrees C lower and the van't Hoff enthalpy difference delta HvH for the helix-coil transition is reduced by 20% (in NaCl) and 10% (in MgCl2) compared to that of ps-D1.D2. Based on energy minimizations of a ps-[d(T5GA5).d(A5CT5)] duplex using force field calculations we propose a model for the conformation of a trans dG.dC base pair in a ps helix.