Duplex formation and the onset of helicity in poly d(CG)n oligonucleotides in a solvent-free environment.

The gas-phase conformations of a series of cytosine/guanine DNA duplexes were examined by ion mobility and molecular dynamics methods. Deprotonated duplex ions were formed by electrospray ionization, and their collision cross sections measured in helium were compared to calculated cross sections of theoretical models generated by molecular dynamics. The 4-mer (dCGCG) and 6-mer (dCGCGCG) duplexes were found to have globular conformations. Globular and helical structures were observed for the 8-mer (dCGCGCGCG) duplex, with the globular form being the more favored conformer. For the 10-mer (dCGCGCGCGCG), 14-mer (dCGCGCGCGCGCGCG), and 18-mer (dCGCGCGCGCGCGCGCGCG) duplexes, only helical structures were observed in the ion mobility measurements. Theory predicts that the helical structures are less stable than the globular forms in the gas phase and should collapse into the globular form given enough time. However, molecular dynamics simulations at 300 K indicate the helical structures are stable in aqueous solution and will retain their conformations for a limited time in the gas phase. The presence of helical structures in the ion mobility experiments indicates that the duplexes retain "solution structures" in the gas phase on the millisecond time scale.

Template-dependent biosynthesis of poly(G) x poly (C) and its antiviral activity in vitro and in vivo.

Experimental conditions for poly(G) synthesis from GTP on a poly(C) template with the aid of Escherichia coli DNA-dependent RNA polymerase were investigated. The reaction product was purified without the use of RNase. On the basis of spectral data, gel permeation chromatography, affinity adsorption and electron microscopic visualization, the poly(G) x poly(C) product was assumed to possess a high degree of structural regularity. Its in vitro and in vivo antiviral activities were compared with those of traditional poly(G) x poly(C) and poly(I) x poly(C). Template-dependent poly(G) x poly(C) was similar in its in vitro activity to poly(I) x poly(C) or even surpassed it, whereas the 'traditional' poly(G) x poly(C) was only slightly active in vitro. However, 'traditional' poly(G) x poly(C) and poly(I) x poly(C) had similar activity in vivo, whereas template-dependent poly(G) x poly(C) was much less active in vivo. The role of intramolecular structural regularity in the in vitro and in vivo antiviral activity of polyribonucleotide duplexes is discussed.

Synthesis of long prebiotic oligomers on mineral surfaces.

Most theories of the origin of biological organization assume that polymers with lengths in the range of 30-60 monomers are needed to make a genetic system viable. But it has not proved possible to synthesize plausibly prebiotic polymers this long by condensation in aqueous solution, because hydrolysis competes with polymerization. The potential of mineral surfaces to facilitate prebiotic polymerization was pointed out long ago. Here we describe a system that models prebiotic polymerization by the oligomerization of activated monomers--both nucleotides and amino acids. We find that whereas the reactions in solution produce only short oligomers (the longest typically being a 10-mer), the presence of mineral surfaces (montmorillonite for nucleotides, illite and hydroxylapatite for amino acids) induces the formation of oligomers up to 55 monomers long. These are formed by successive 'feedings' with the monomers; polymerization takes place on the mineral surfaces in a manner akin to solid-phase synthesis of biopolymers.

Crystal structure of the highly distorted chimeric decamer r(C)d(CGGCGCCG)r(G).spermine complex--spermine binding to phosphate only and minor groove tertiary base-pairing.

The crystal structure of the self-complementary chimeric decamer duplex r(C)d(CGGCGCCG)r(G), with RNA base pairs at both termini, has been solved at 1.9 A resolution by the molecular replacement method and refined to an R value of 0.145 for 2,314 reflections. The C3'-endo sugar puckers of the terminal riboses apparently drive the entire chimeric duplex into an A-DNA conformation, in contrast to the B-DNA conformation adopted by the all-deoxy decamer of the same sequence. Five symmetry related duplexes encapsulate a spermine molecule which interacts with ten phosphate groups, both directly and through water molecules to form multiple ionic and hydrogen bonding interactions. The spermine interaction severely bends the duplexes by 31 degrees into the major groove at the fourth base pair G(4).C(17), jolts it and slides the 'base plate' into the minor groove. This base pair, together with the adjacent base pair in the top half and the corresponding pseudo two-fold related base pairs in the bottom half, form four minor groove base-paired multiples with the terminal base pairs of two neighboring duplexes.

Preparation and duplex-to-single strand transition of the fully complementary duplex of d(G4).d(C4) in aqueous solution.

The d(G4) and d(C4) molecules in the single stranded state were synthesized by the phosphotriester method and purified. The full duplex of tetramer d(G4).d(C4) was prepared by expending about a month. The duplex-to-single strand transition was observed by UV-spectroscopy. A standard hypochromic effect was observed, which is different from some experimental results reported previously.

[Dependence of the antiviral activity of the poly(G).poly(C) complex on the size of the continuous poly(C)segments]. / Zavisimost' protivovirusnoi aktivnosti kompleksa poli(G).poli(Ts) ot razmera nepreryvnykh uchastkov poli(Ts).

Modification of poly(C) by various frequency treatment with adenosine non-complementary to guanosine has produced poly(G) X poly (C.A) complexes with continuous double-stranded areas the length of which is determined by C/A ratio. Studies of the antiviral activity of poly(G).poly(C,A) complexes with C/A from 10:1 to 90:1 and poly(G).poly(C) in vesicular stomatitis virus-infected chick embryo cell cultures and in experimental tick-borne encephalitis of mice demonstrated that the maximum activity is achieved at an average lengths of double-stranded areas of 90 nucleotide pairs. At the same time, a low but statistically significant antiviral activity is observed at a length of double-stranded areas of 10-30 nucleotide pairs.

Complexes of polyriboguanylate with modified polyribocytidylate.

It was established minimal length of continuous poly(C) sequence of poly(G).poly(C) complex required for effective interferon induction by investigation poly(G).poly(C,A) with different molar ratios of C:A varying from 10:1 to 90:1. The minimum length of the double-stranded sequence of the macromolecule complex poly(G).poly(C) is equal to at least 90-100 nucleotides. The effect of 5-halogen-polyribocytidilates on the properties of the complexes has been also investigated.

Preparation and biological properties of a highly active poly(G) X poly(C) inducer of interferon.

Experimental conditions necessary for the full expression of interferon-inducing activity by a complex of polyguanylic acid and polycytidylic acid included: (i) a sufficiently high molecular size of each homopolymer; (ii) annealing conditions which insured complete denaturation of polyguanylic acid self-structure; and (iii) the specific biological assay employed to assay interferon-inducing potency. The complex of polyguanylic acid and polycytidylic acid possessed several properties that suggested it may be an atypical polynucleotide interferon inducer. For instance, it was inactive in primary rabbit kidney cell cultures, usually exquisitely sensitive to polynucleotide interferon inducers, unless it was incubated on the cell cultures for prolonged times or in the presence of DEAE-dextran. Polyguanylic acid X polycytidylic acid could induce interferon in rabbits and mice but gave a more protracted response than did poly(I) X poly(C). Finally, poly(G) X poly(C) was, without any modification, resistant to degradation by serum nucleases.

Poly(7-deazaguanylic acid), the homopolynucleotide of the parent nucleoside of queuosine.

Poly(7-deazaguanylic acid) was enzymatically synthesized by the polymerization of 7-deazaguanosine 5'-diphosphate with polynucleotide phosphorylase from Micrococcus luteus in high yield. The homopolymer showed a similar thermal and total hypochromicity to poly(G) at the long wavelength absorption maximum. No sigmoid melting profile was observed for poly(c7G) as is found for poly(G), implying a single-stranded structure in aqueous solution. From the circular dichroism spectra it can be concluded that the 7-deazapurine nucleotide is much more flexible than the purine nucleotide. In analogy to poly(G), the homopolymer poly(c7G) forms a 1:1 complex with poly(C) under neutral conditions, melting at a similar temperature to the poly(G) complex. However, at pH 2.5, where a poly(G) X 2poly(C) complex is observed, poly(c7G) still binds only one poly(C) strand. This is due to the lack of N-7 in poly(c7G), not allowing Hoogsteen base pair formation, which occurs with poly(G). RNase T1 cleaves poly(c7G), indicating that N-7 of guanosine is not a requirement for nucleotide binding to the enzyme, as has been suggested. Because of the single-stranded structure of poly(c7G), the polynucleotide chain is rapidly hydrolyzed by the single-strand-specific nuclease S1, whereas multistranded poly(G) is completely resistant.