Transition-metal complexes as inhibitors of proteins recognizing double-stranded fragments of nucleic acids.

A 30-membered DNA duplex containing the recognition site of restriction endonuclease SsoII and a 30-membered RNA--DNA hybrid duplex, a substrate of E. coli RNase H, were synthesized. The cleavage of the 30-membered fragments of nucleic acids catalyzed by endonucleases in the presence of Co-phthalocyanine complex [CoPc(COONa)8] containing eight carboxyl groups at the periphery of the ligand was studied. It was shown that the efficiency of enzyme catalysis decreases in the presence of the metal complex for both endonucleases. By addition of a 100-fold excess of Co-phthalocyanine complex with respect to DNA duplex the initial rate of substrate hydrolysis by restriction endonuclease SsoII is observed to decrease twice. An equimolar ratio of the metal complex and hybrid duplex leads to essentially complete inhibition of RNA cleavage by RNase H from E. coli. The inhibition of catalytic activity of enzymes recognizing the double-stranded nucleic acids in the presence of Co-phthalocyanine complex is assumed to be caused by the ability of the latter to interact with DNA, RNA, and DNA--RNA duplexes.

Use of UV spectroscopy for the study of nucleic acid cleavage by E. coli RNase H and restriction endonucleases.

A one-step spectrophotometric method for monitoring of nucleic acid cleavage by ribonuclease H from E. coli and type II restriction endonucleases has been proposed. It is based on recording of the increase in the UV absorbance at 260 nm during the course of enzymatic reaction. Duplexes stable under the reaction conditions were chosen as substrates for the enzymes being studied. In order to obtain duplex dissociation following their cleavage by the enzyme appreciate temperature conditions were selected. The spectrophotometric method may be applied for rapid testing of the nuclease activity in protein preparations as well as for precise quantitative analysis of nucleic acid degradation by enzymes. This method may be successfully employed in kinetic studies of nucleic acid-protein interactions.

Oligonucleotide circularization by template-directed chemical ligation.

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), hairpin-like 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.

The use of oligonucleotide probes containing 2'-deoxy-2'-fluoronucleosides for regiospecific cleavage of RNA by RNase H from Escherichia coli.

Protected 2'-deoxy-2'-fluorouridine and 2'-deoxy-2'-fluorocytidine suitable for incorporation into oligonucleotides via the phosphoramidite approach have been prepared. Five modified and two unmodified oligonucleotides have been synthesized to investigate the regiospecific cleavage of a 5S RNA from Escherichia coli by RNase H. In order to show whether the modified oligonucleotides are able to hybridize with the RNA the physico-chemical properties (melting curves, CD spectra) of analogous DNA/oligodeoxyribonucleotide duplexes have been examined. The modified oligonucleotides are shown to form stable duplexes with a DNA-matrix which exist in an A-like form. Two of the modified probes containing four 2'-deoxy-2'-fluorocytidines or two 2'-deoxy-2'-fluorouridines direct the splitting by RNase H of only one phosphodiester bond of the RNA.

Synthesis of oligonucleotide probes containing 2'-deoxy-2'-fluoronucleosides for cleavage of RNA by RNase H.

Modified oligodeoxyribonucleotides containing 3'-terminal 2'-deoxy-2'-fluorouridine (UF) or 2'-fluorothymidine (TF) were successfully applied for specific RNA hydrolysis by RNase H from E. coli. The nonanucleotides d(CACCGCGCTF) and d(CACCGCGCUF) were synthesized using the phosphoramidite solid support method. The modified nucleosides were immobilized on the CPG support and provided the starting nucleoside residues. Model experiments were carried out using the 5S RNA from E. coli ribosomes and its 1-41 fragment. It was found that the use of this type of modified probes did not decrease neither the efficiency nor the specificity of the RNase H reaction.

Influence of probe structure on unique (regiospecific) cleavage of RNA by RNase H.

Chimeric oligo(ribo-deoxyribo)nucleotides with an internucleotide pyrophosphate bond are novel probes for regiospecific hydrolysis of RNA by RNase H. It has been shown that the use of d(TGTGTAT)ppGCCAU leads to unique hydrolysis of the TMV RNA fragment pAAUGGCAUACAC between C10 and A11.