Modified dinucleoside tetraphosphonates, new potential inhibitors of HIV reverse transcriptase.

New gamma-substituted analogues of dNTP were synthesized and their enzymatic stability and antiviral properties were evaluated.

Analysis of poly(A)+RNA patterns in human tissues.

A novel method for analysing and comparing the relative amounts of the most abundant (higher than 0.1% abundance) individual mRNAs present in different poly(A)+RNA preparations has been developed. This method is based on the synthesis of short (10-30 nucleotide) cDNA termination products, by reverse transcription of poly(A)+RNA primed with a 5'-labeled oligonucleotide. A set of 30 different oligonucleotides are used as primers in separate reactions, their length and sequences having been chosen to provide more than a 90% probability of initiating synthesis from any individual RNA present in the poly(A)+RNA. Each primer produces about 10-60 bands per track, following polyacrylamide gel electrophoresis under denaturing conditions. Data presented reveals poly(A)+RNA pattern differences for a number of human tissues and identifies changes in RNA patterns between normal tissues and neoplastic human tumors (myoma of the uterus) from several individuals.

3'-Fluoro-3'-deoxyribonucleoside 5'-triphosphates: synthesis and use as terminators of RNA biosynthesis.

3'-Fluoro-3'-deoxy-uridine, -cytidine, -adenosine and -guanosine have been synthesized by glycosylation of the corresponding silylated bases with 1-O-acetyl-2,5-di-O-benzoyl-3-fluoro-3-deoxy-D-ribofuranose in the presence of Friedel-Crafts catalysts and were converted to the 5'-triphosphates, NTP(3'-F). It was shown that NTP(3'-F) are terminators of RNA synthesis catalyzed by DNA-dependent RNA polymerase from E. coli and may thus serve as tools for DNA sequencing.

The influence of a double-stranded hindrance on DNA synthesis performed by DNA polymerase alpha, T4 DNA polymerase, DNA polymerase I (Klenow fragment) and AMV reverse transcriptase.

The influence of a double-stranded region on DNA synthesis performed by a series of DNA polymerases on a single-stranded template was studied. Two types of double-stranded hindrances were employed: a stable hairpin formed by the template alone and a region formed by the template and an extraneous oligonucleotide complementary to the template. While T4 and calf thymus alpha DNA polymerases are strongly arrested at the beginning of either of the two double-stranded hindrances, the Klenow fragment of E. coli DNA polymerase I and avian myeloblastosis virus reverse transcriptase can pass through the double-stranded regions. The DNA chain-elongation rate seems to be undisturbed in the case of reverse transcriptase but greatly reduced for DNA polymerase I.

Nucleoside 5'-triphosphates modified at sugar residues as substrates for calf thymus terminal deoxynucleotidyl transferase and for AMV reverse transcriptase.

Terminal deoxynucleotidyl transferase from calf thymus and RNA-directed DNA polymerase (reverse transcriptase) from the avian myeloblastosis virus catalyze the incorporation of 3'-amino-2',3'-dideoxynucleoside 5'-triphosphates, as well as some of their 3'-derivatives, 3'-amino-3'-deoxyarabinonucleoside 5'-triphosphates and some other nucleoside 5'-triphosphates modified at sugar residues. After incorporation of the appropriate 5'-mononucleotide residue into the DNA, further chain elongation is blocked. This finding opens up a possibility for selective inhibition of DNA synthesis catalyzed by a certain enzyme.

3'-Hydroxymethyl 2'-deoxynucleoside 5'-triphosphates are inhibitors highly specific for reverse transcriptase.

dNTP(3'-OCH3), a 3'-O-methyl derivative of dNTP, is a chain terminator substrate for DNA synthesis catalyzed by AMV reverse transcriptase. The enzyme seems to be the only DNA polymerase susceptible to the inhibitor while all the other DNA polymerases tested are fully resistant to the nucleotide analog. The resistant polymerases are: E. coli DNA polymerase I, Klenow's fragment of DNA polymerase I, phage T4 DNA polymerase, calf thymus DNA polymerase alpha, rat liver DNA polymerase beta and calf thymus terminal deoxyribonucleotidyl transferase.

3'-Fluoro-2',3'-dideoxyribonucleoside 5'-triphosphates: terminators of DNA synthesis.

It is shown that dNTP(3'F) are terminators of DNA synthesis and may serve as very effective tools for DNA sequencing with E.coli DNA polymerase I and AMV reverse transcriptase. The dNTP(3'F) are found to be chain terminator substrates for calf thymus terminal deoxyribonucleotidyl transferase but not for calf thymus DNA polymerase alpha. The optimal dNTP(3'F) concentration for DNA sequencing by DNA polymerase I is found to be an order of magnitude lower than that of ddNTPs. dNTP(3'F) produce a more clear sequence pattern than do ddNTPs.

2',3'-Dideoxy-3' aminonucleoside 5'-triphosphates are the terminators of DNA synthesis catalyzed by DNA polymerases.

It is shown that 2',3'-dideoxy-3'-aminonucleoside 5'-triphosphates with adenine, guanine, cytosine and thymine bases are effective inhibitors of DNA polymerase I, calf thymus DNA polymerase alpha and rat liver DNA polymerase beta. The effect of the above-mentioned compounds is markedly higher than corresponding action of the well-known DNA synthesis inhibitors arabinonucleoside 5'-triphosphates and 2',3'-dideoxynucleoside 5'-triphosphates. 2',3'-dideoxy-3'-aminonucleoside 5'-monophosphate residues incorporate into the 3'-terminus of the primer and terminate the DNA chain elongation. The possibility of using 2',3'-dideoxy-3'-aminonucleoside 5'-triphosphates as terminators for DNA sequencing by the polymerization method is demonstrated.

Binding of actinomycin D to DNA revealed by DNase I footprinting.

We have analyzed the specificity of the actinomycin D-DNA interaction. The 'footprint' method has been used in this investigation. It is shown that: (i) The presence of dinucleotide GC or GG is required for binding of a single drug molecule. (ii) The strong binding sites are encoded by tetranucleotide XGCY; where X not equal to G and Y not equal to C in accordance with RNA elongation hindrance sites [1]. (iii) There is a positive cooperativity in binding of actinomycin D with DNA.

Sequence-specific inhibition of RNA elongation by actinomycin D.

We have developed a method to localize specific sites where RNA elongation is arrested due to DNA-bound ligands. The method was used to determine apparent binding sites for actinomycin D. We have found 14 strong RNA hindrance sites along nucleotide sequence of T7 and D111 T7 DNA of 380 nucleotides full length under low actinomycin D concentration conditions. Nucleotide sequence of all the sites is described by general formula XGCY where X not equal to G and Y not equal to C.

Stability of cloned promoter-containing fragments.

Strong bacteriophages lambda and T7 promoters for Escherichia coli RNA polymerase were cloned in a multicopy plasmid. To achieve this result, two variants of the promoter-probe vectors were constructed. It was found that (i) modifications of the nucleotide sequence, apart from the commonly accepted promoter region, both upstream and downstream of the RNA initiation point greatly influenced the efficiency of promoters in vivo, (ii) a recombinant DNA composed of one of the promoter-probe plasmids and a tandem of A1, A2, and A3 promoters of T7 bacteriophage DNA induced a reproducible secondary change in plasmid DNA upon cloning. This change was substitution of the part of the recombinant that originated as T7 by a large portion of the host DNA.

Investigation of the binding of Escherichia coli RNA polymerase to DNA from bacteriophages T2 and T7 by kinetic formaldehyde method and electron microscopy.

The complexes of T2 DNA with RNA polymerase of Escherichia coli were studied by two methods: kinetic formaldehyde method with preliminary fixation of complexes with low formaldehyde concentrations, and electron microscopy. For electron-microscopic investigations the effect of different conditions of formaldehyde fixation for DNA-RNA-polymerase complexes was studied and optimal fixation conditions were found. The suggested fixation method for DNA-RNA-polymerase complexes allows investigation of RNA polymerase molecule distribution on DNA in a wide range of conditions (ionic strength of the solution, weight ration of enzyme to DNA etc.). The comparison of the concentration of RNA polymerase molecules bound to DNA, determined by electron microscopy, and the concentration of defects in DNA as determined by the kinetic formaldehyde method, showed their coincidence. The electron-microscopic procedure was used to make maps of RNA polymerase distribution on T7 DNA. A correlation between the binding regions of the enzyme and the genetic map of early DNA T7 region was found.

The study of DNA-RNA-polymerase complexes by kinetic formaldehyde method.

A modification of the kinetic formaldehyde method has been proposed providing a possibility for locally denatured regions (defects) formed in DNA preincubated with RNA polymerase (in the absence of nucleoside triphosphates) to be detected. This modification consists in a previous fixation of DNA-enzyme complex with small concentrations of formaldehyde, which do not induce formation of defects in DNA alone. The method has been calibrated under the conditions favourable to RNA synthesis. Studies of the effect of the fixation conditions on the number of defects in DNA interacting with RNA polymerase have shown that the number of defects is constant with formaldehyde fixation concentration between 0.05% and 0.3-0.5% and with fixation time between 2 min and 100 min. The dependence of the number of defects in DNA on RNA polymerase concentration at low ionic strength (0.05 M KCl) is presented by a curve with a plateau. From the initial linear part of the curve it has been found that the enzyme bound to DNA as a monomer. At the excess of the enzyme the mean number of nucleotide pairs between defects is 400-500. Increase of ionic strength results in decrease of the number of defects in DNA. The number of defects depends on temperature of preincubation of the complex. There were no defects in DNA at temperatures below 20 degrees C. At temperatures above 30 degrees C the number of defects reaches saturation. A sharp transition occurs in the range of temperatures between 20 degrees C and 30 degrees C. Analysis of the experimental and literature data, concerning the interaction of formaldehyde and amino acid methylol derivatives with DNA bases, leads to the conclusion that the mechanism of the formation of defects in helical DNA most likely consists in its unwinding or sharp weakening upon binding of RNA polymerase, prior to addition of formaldehyde.