Duplex stability of DNA.DNA and DNA.RNA duplexes containing 3'-S-phosphorothiolate linkages

3'-S-Phosphorothiolate (3'-SP) linkages have been incorporated into the DNA strand of both a DNA.RNA duplex and a DNA.DNA duplex. Thermal melting (T(m)) studies established that this modification significantly stabilises the DNA.RNA duplex with an average increase in T(m) of about 1.4 degrees C per modification. For two or three modifications, the increase in T(m) was larger for an alternating, as compared to the contiguous, arrangement. For more than three modifications their arrangement had no effect on T(m). In contrast to the DNA.RNA duplex, the 3'-S-phosphorothiolate linkage destabilised the DNA.DNA duplex, irrespective of the arrangement of the 3'-SP linkages. The effect of ionic strength on duplex stability was similar for both the phosphorothiolate-substituted and the unmodified RNA.DNA duplexes. The results are discussed in terms of the influence that the sulfur atom has on the conformation of the furanose ring and comparisons are also drawn between the current study and those previously conducted with other modifications that have a similar conformational effect.     

Synthesis of dinucleotides containing nitrone, hydroxylamine and amidoxime linkages

Three new thymidine dinucleotides with nitrone, hydroxylamine and amidoxime backbone linkages, suitable for incorporation into oligonucleotide chains, were easily synthesized, by coupling of readily available thymidine monomers and applying short, simple and efficient procedures.     

Efficient conjugation and preferential DNA binding of oligonucleotides containing 2′-O-(2-oxoethyl)arabinouridine

Oligodeoxyribonucleotides were synthesized that contain a novel nucleoside, 2′-O-(2-oxoethyl)arabinouridine. Whereas such oligonucleotides showed only a slight reduction in the T_M values of their complexes with complementary DNA, a significant destabilization was observed in the case of duplexes formed with RNA. This may be explained by the C_2′-endo conformation of 2′-O-(2,3-dihydroxypropyl)arabinouridine as demonstrated by NMR experiments in D₂O. The modified oligonucleotides were used to synthesize a number of conjugates with dyes, biotin and a N-modified laminin peptide, by hydrazone and oxime formation. We suggest that the 2′-arabinoaldehyde-containing DNA duplexes may be valuable tools for affinity modification of DNA-binding proteins.     

NMR solution structure of an oligonucleotide hairpin with a 2'F-ANA/RNA stem: implications for RNase H specificity toward DNA/RNA hybrid duplexes.

The first structure of a 2'-deoxy-2'-fluoro-D-arabinose nucleic acid (2'F-ANA)/RNA duplex is presented. We report the structural characterization by NMR spectroscopy of a small hybrid hairpin, r(GGAC)d(TTCG)2'F-a(GTCC), containing a 2'F-ANA/RNA stem and a four-residue DNA loop. Complete (1)H, (13)C, (19)F, and (31)P resonance assignments, scalar coupling constants, and NOE constraints were obtained from homonuclear and heteronuclear 2D spectra. In the chimeric duplex, the RNA strand adopts a classic A-form structure having C3' endo sugar puckers. The 2'F-ANA strand is neither A-form nor B-form and contains O4' endo sugar puckers. This contrasts strongly with the dynamic sugar conformations previously observed in the DNA strands of DNA/RNA hybrid duplexes. Structural parameters for the duplex, such as minor groove width, x-displacement, and inclination, were intermediate between those of A-form and B-form duplexes and similar to those of DNA/RNA duplexes. These results rationalize the enhanced stability of 2'F-ANA/RNA duplexes and their ability to elicit RNase H activity. The results are relevant for the design of new antisense drugs based on sugar-modified nucleic acids.     

Synthesis and triplex-forming properties of oligonucleotides containing thio-substituted C-nucleoside 4-thiopseudoisocytidine

We synthesized the 3′-phosphoramidite building block of 4-thiopseudoisocytidine (s⁴ΨiC) and incorporated it into triplex-forming oligonucleotides (TFOs). The results of thermal denaturation of triplexes incorporating s⁴ΨiC showed that s⁴ΨiC could be used as a nucleoside component of TFOs to increase the thermal stability of triplexes at pH 7.     

Chimeric RNA Oligonucleotides with Triazole and Phosphate Linkages: Synthesis and RNA Interference

Chimeric RNA oligonucleotides with an artificial triazole linker were synthesized using solution‐phase click chemistry and solid‐phase automated synthesis. Scalable synthesis methods for jointing units for the chimeric structure have been developed, and after click‐coupling of the jointing units with triazole linkers, a series of chimeric oligonucleotides was prepared by utilizing the well‐established phosphoramidite method for the elongation. The series of chimeric 21‐mer oligonucleotides that possessed the triazole linker at different strands and positions allowed for a screening study of the RNA interference to clarify the preference of the triazole modifications in small‐interfering RNA molecules.     

GA and AG sequences of DNA react with cisplatin at comparable rates

The sequence selectivity of the antitumor drug cisplatin (cis-[PtCl(2)(NH(3))(2)] (1)) between the 5'-AG-3' and 5'-GA-3' sites of DNA has been a matter of discussion for more than twenty years. In this work, we compared the reactivity of GA and AG sequences of DNA towards the aquated forms of cisplatin (cis-[PtCl(NH(3))(2)(H(2)O)](+) (2), cis-[Pt(NH(3))(2)(H(2)O)(2)](2+) (3), and cis-[Pt(OH)(NH(3))(2)(H(2)O)](+) (4)) using two sets of experiments. In the first, we investigated a DNA hairpin, whose duplex stem contained a TGAT sequence as the single reactive site, and determined the individual rate constants of platination with 2 and 3 for G and A in acidic solution. The rate constants at 20 degrees C in 0.1M NaClO(4) at pH 4.5+/-0.1 were 0.09(4) M(-1)s(-1) (G) and 0.11(3) M(-1)s(-1) (A) for 2, and 9.6(1) M(-1)s(-1) (G) and 1.7(1) M(-1)s(-1) (A) for 3. These values are similar to those obtained previously for an analogous hairpin that contained a TAGT sequence. The monoadducts formed with 2 by both GA purines are extremely long-lived, partly as a result of the slow hydrolysis of the chloro monoadduct at A, and partly because of the very low chelation rate (1.4 x 10(-5)s(-1) at 20 degrees C) of the aqua monoadduct on the guanine. In the second set of experiments, we incubated pure or enriched samples of 1, 2, 3, or 4 for 18-64 h at 25 degrees C with a 19 base pair (bp) DNA duplex, whose radiolabeled top strand contained one GA and one AG sequence as the only reactive sites. Quantification of the number of GA and AG cross-links afforded a ratio of about two in favor of AG, irrespective of the nature of the leaving ligands. These results disagree with a previous NMR spectroscopy study, and indicate that GA sequences of DNA are substantially more susceptible to attack by cisplatin than previously thought.     

Exploring concerted effects of base pairing and stacking on the excited‐state nature of DNA oligonucleotides by DFT and TD‐DFT studies

We have taken (dA)₅, (dT)₅, and (dA)₅·(dT)₅ as model systems to study concerted effects of base pairing and stacking on excited‐state nature of DNA oligonucleotides using density functional theory (DFT) and time dependent DFT methods. The spectroscopic states are determined to be of a partial A → A charge‐transfer nature in the A·T oligonucleotides. The T → T charge‐transfer transitions produce dark states, which are hidden in the energy region of the steady‐state absorption spectra. This is different from the previous assignment that the T → T charge‐transfer transition is responsible for a shoulder at the red side of the first strong absorption band. The A → T charge‐transfer states were predicted to have relatively high energies in the A·T oligonucleotides. The present calculations predict that the T → A charge‐transfer states are not involved in the spectra and excited‐state dynamics of the A·T oligonucleotides. In addition, the influence of base pairing and stacking on the nature of the ¹nπ* and ¹ππ* states are discussed in detail. © 2010 Wiley Periodicals, Inc. Int J Quantum Chem, 2011     

Aminoglycoside complexation with a DNA.RNA hybrid duplex: the thermodynamics of recognition and inhibition of RNA processing enzymes

Spectroscopic and calorimetric techniques were employed to characterize and contrast the binding of the aminoglycoside paromomycin to three octamer nucleic acid duplexes of identical sequence but different strand composition (a DNA.RNA hybrid duplex and the corresponding DNA.DNA and RNA.RNA duplexes). In addition, the impact of paromomycin binding on both RNase H- and RNase A-mediated cleavage of the RNA strand in the DNA.RNA duplex was also determined. Our results reveal the following significant features: (i) Paromomycin binding enhances the thermal stabilities of the RNA.RNA and DNA.RNA duplexes to similar extents, with this thermal enhancement being substantially greater in magnitude than that of the DNA.DNA duplex. (ii) Paromomycin binding to the DNA.RNA hybrid duplex induces CD changes consistent with a shift from an A-like to a more canonical A-conformation. (iii) Paromomycin binding to all three octamer duplexes is linked to the uptake of a similar number of protons, with the magnitude of this number being dependent on pH. (iv) The affinity of paromomycin for the three host duplexes follows the hierarchy, RNA.RNA > DNA.RNA > DNA.DNA. (v) The observed affinity of paromomycin for the RNA.RNA and DNA.RNA duplexes decreases with increasing pH. (vi) The binding of paromomycin to the DNA.RNA hybrid duplex inhibits both RNase H- and RNase A-mediated cleavage of the RNA strand. We discuss the implications of our combined results with regard to the specific targeting of DNA.RNA hybrid duplex domains and potential antiretroviral applications.     

Functional tuning of nucleic acids by chemical modifications: tailored oligonucleotides as drugs,devices, and diagnostics

Chemical modifications of nucleic acids present vast opportunities for extending the functions and properties of these biomolecules. In general, efforts invested in this direction pertain to the introduction of reactive functional groups for further derivatizations of oligonucleotides with numerous reporter groups and for equipping nucleic acids with catalytic chemical moieties. This review deals with representative chemical modifications in the nucleobases, sugars, and the phosphate ester backbone and their application from novel catalytic RNA selection to nucleic acid-based biosensors.     

Synthesis of 2′-hydrazine oligonucleotides and their efficient conjugation with aldehydes and 1,3-diketones

Oligodeoxyribonucleotides that contain a novel nucleoside, 2′-O-(2-hydrazinoethyl)uridine, were synthesised by NaBH₃CN reduction of hydrazones formed from 2′-O-(2-oxoethyl)oligonucleotides with FmocNHNH₂, followed by concd aq NH₃ deprotection. The 2′-hydrazine oligonucleotides obtained were then used to synthesise a number of conjugates with aldehydes via hydrazone formation and with1,3-diketones via pyrazole formation. The method was shown to be applicable for the preparation of oligonucleotide-peptide conjugates.     

Hoogsteen-Duplex DNA: Synthesis and base pairing of oligonucleotides containing 1-deaza-2′-deoxyadenosine

Oligodeoxyribonucleotides containing 1-deaza-2′-deoxyadenosine ( = 7-amino-3-(2-deoxy-β-D -erythro-pentofuranosyl)-3H-imidazo[4, 5-b]pyridine; 1b ) form Hoogsteen duplexes. Watson-Crick base pairs cannot be built up due to the absence of N(1). For these studies, oligonucleotide building blocks – the phosphonate 3a and the phosphoramidite 3b – were prepared from 1b via 4a and 5 , as well as the Fractosil-linked 6b , and used in solid-phase synthesis. The applicability of various N-protecting groups (see 4a – c ) was also studied. The Hoogsteen duplex d[(c¹A)₂₀] · d(T₂₀) ( 11 · 13 ; T_m 15°) is less stable than d(A₂₀) · d(T₂₀) ( 12 · 13 ; T_m 60°). The block oligomers d([c¹A)₁₀–;T₁₀] ( 14 ) and d[T₁₀–(c¹A)₁₀] ( 15 ) containing purine and pyrimidine bases in the same strand are also able to form duplexes with each other. The chain polarity was found to be parallel.     

Phosphoramidites and solid supports based on N-substituted 2,4-dihydroxybutyramides: universal reagents for synthesis of modified oligonucleotides

A general and convenient method for synthesis of modified oligonucleotides by use of new non-nucleoside phosphoramidites is reported. A chiral 1,3-diol backbone of the modifying reagents is generated either from (R)-(+)-α-hydroxy-γ-butyrolactone or (R)-(−)-pantolactone. Aliphatic amines were acylated with the lactones to give the corresponding N-substituted 2,4-dihydroxybutyramides. After protection of a side chain, if necessary, the diols were converted into phosphoramidites or solid supports suitable for use in oligonucleotide synthesis. The reagents allow single, multiple or combined introduction of various functions (e.g., alkylamine, imidazole and pyrene residues) into synthetic oligonucleotides. The structures of the conjugates were confirmed by MALDI-TOF mass spectrometry.