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Here we investigated the ability of the human X-family DNA polymerases β and λ to bypass thymine glycol (Tg) in gapped DNA substrates with the damage located in a defined position of the template strand. Maximum velocities and the Michaelis constant values were determined to study DNA synthesis in the presence of either Mg2+or Mn2+. Additionally, the influence of hRPA (human replication protein A) and hPCNA (human proliferating cell nuclear antigen) on TLS (translesion synthesis) activity of DNA polymerases β and λ was examined. The results show that (i) DNA polymerase λ is able to catalyze DNA synthesis across Tg, (ii) the ability of DNA polymerase λ to elongate from a base paired to a Tg lesion is influenced by the size of the DNA gap, (iii) hPCNA increases the fidelity of Tg bypass and does not influence normal DNA synthesis catalyzed by DNA polymerase λ, (iv) DNA polymerase β catalyzes the incorporation of all four dNTPs opposite Tg, and (v) hPCNA as well as hRPA has no specific effect on TLS in comparison with the normal DNA synthesis catalyzed by DNA polymerase β. These results considerably extend our knowledge concerning the ability of specialized DNA polymerases to cope with a very common DNA lesion such as Tg.

Bacteria synthesize small regulatory RNAs known as 6S RNAs which bind to RNA polymerase and regulate transcription. This paper describes an analysis of two 6S RNAs produced in Bacillus subtilis. Although these two RNAs share many features, there are also distinct differences, the mechanistic implications of which are discussed.

This review outlines the design and synthesis of oligonucleotides bearing 2'-modified residues at predetermined positions within the strand. The relative merits of incorporation of reactive carboxyl, carbonyl, iodoacetamide and disulfide-containing groups into oligonucleotides were considered along with solid-phase synthesis of DNA 2'- conjugates. The specific cross-linking of 2'-substituted oligonucleotides to nucleic acid-binding proteins (transcription factor NF-κB, restriction-modification enzymes) was shown to be helpful in scanning the protein-DNA interface and studying the conformational dynamics of biopolymer ensembles. The future perspectives of chemically reactive DNA constructs as specific protein decoys are discussed.

A simple and rapid approach for detecting apurinic (AP) sites in DNA, based on direct stripping chronopotentiometric measurements of the adenine and guanine nucleobases at a graphite electrode is described. Tetrahydrofuranyl residues, lacking a nucleobase moiety, were utilized for designing the AP sites and were incorporated in 19‐mer oligonucleotides. The change of adenine‐to‐guanine response ratio (A/G) in one‐, two‐ or three‐substituted adenosine residues for stable analogs of AP sites was exploited for electrochemical measurements of the adenine loss. The resulting A/G response ratio decreases linearly upon increasing the number of AP sites in the oligonucleotides; the values of A/G electrochemical signals were slightly enhanced when compared to the actual purine content. HPLC analysis of the released nucleobases confirmed that the sulfuric acid‐induced oligonucleotide cleavage provides complete apurination and dissolution of the released nucleobases in aqueous solution. Additional experiments with mixtures of free nucleobases and purine nucleosides reveal that the larger A/G ratio observed in the electrochemical analysis of AP‐site‐containing oligomers is attributed to the influence of the acid and/or thermal decomposition products (particularly the sugar fragments). This study represents the first step in developing a simple and direct electrochemical assay of AP sites in single‐stranded DNA.

An efficient method for synthesis of oligonucleotide 5'-conjugates through amide-bond formation on solid phase is described. Protected oligonucleotides containing a 5'-carboxylic acid function were obtained by use of a novel non-nucleosidic phosphoramidite building block, where the carboxylic acid moiety was protected by a 2-chlorotrityl group. The protecting group is stable to the phosphoramidite coupling conditions used in solid-phase oligonucleotide assembly, but is easily deprotected by mild acidic treatment. The protecting group may be removed also by ammonolysis. 5'-Carboxylate-modified oligonucleotides were efficiently conjugated on solid support under normal peptide-coupling conditions to various amines or to the N-termini of small peptides to yield products of high purity. The method is well-suited in principle for the synthesis of peptide-oligonucleotide conjugates containing an amide linkage between the 5'-end of an oligonucleotide and the N-terminus of a peptide.

Oligonucleotide‐peptide conjugates have several applications, including their potential use as improved antisense agents for interfering with the RNA function within cells. In order to provide robust and generally applicable conjugation chemistry, we developed a novel approach of fragment coupling of pre‐synthesized peptides to the 2′‐position of a selected nucleotide within an otherwise protected oligonucleotide chain attached to a solid support.

Oligonucleotide-peptide conjugates have several applications, including their potential use as improved antisense agents for interfering with the RNA function within cells. In order to provide robust and generally applicable conjugation chemistry, we developed a novel approach of fragment coupling of pre-synthesized peptides to the 2′-position of a selected nucleotide within an otherwise protected oligonucleotide chain attached to a solid support.

The review summarizes published data on the results and achievements in the field of structure and function analysis of protein – nucleic acid complexes by means of main physical and biochemical methods, including X-ray diffraction, nuclear magnetic resonance spectroscopy, electron and atomic force microscopy, small-angle X-ray and neutron scattering, footprinting and cross-linking. Special attention is given to combined approaches. The advantages and limitations of each method are considered, and the prospects of their application for wide-scale structural studies in vivoare discussed.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

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Certain amino acids and short peptides are known to act as enhancers of ribozyme-mediated RNA cleavage at low concentration of magnesium ion. Thus, covalent conjugates of oligonucleotides and some amino acids may have a potential for development as sequence-specific artificial ribonucleases. Here we would like to report an incorporation of basic amino acids, lysine and histidine, into oligonucleotides at the 2′-position of a uridine residue. The approach involves the 2′-<it>O</it>-alkylation and attachment of the corresponding amino acid by the α-amino group through a urethane-type linker.

Oligodeoxyribonucleotides that contain a novel nucleoside, 2′-<it>O</it>-(2-hydrazinoethyl)uridine, were synthesized by sodium cyanoborohydride reduction of hydrazones formed from 2′-aldehyde oligonucleotides and FmocNHNH<inf>2</inf> followed by ammonia deprotection. 2′-Hydrazine oligonucleotides were used successfully to obtain a series of conjugates with various aldehydes by hydrazone formation. The method was shown to be suitable for the preparation of oligonucleotide-peptide conjugates.

For Abstract see ChemInform Abstract in Full Text.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

An efficient method for producing the covalent closure of oligonucleotides on complementary templates by the action of BrCN was developed. A rational design of linear precursor oligonucleotides was studied, and the effect of factors such as oligonucleotide concentration and oligomer-template length ratio was evaluated. The efficiency of circularization was shown to correlate well with the secondary structure of the precursor oligomer (as predicted by a simple computer analysis), hairpinlike structures bearing free termini clearly favouring the circularization reaction. A novel idea, consisting of the incorporation of non-nucleotide insertions in the precursor oligomer (namely, 1,2-dideoxy-D-ribofuranose residues), may render this method universal and highly effective. An original set of assays was developed to confirm the circular structure of the covalently closed oligonucleotides.