Regiospecific inhibition of DNA duplication by antisense phosphate-methylated oligodeoxynucleotides

Abstract

A new synthesis route for long phosphate-methylated oligodeoxynucleotides is described, which were used as antisense inhibitors of the DNA replication. Phosphate-methylated oligomers hybridize more strongly with natural DNA than their natural analogues, due to the absence of electrostatic interstrand repulsions. Compared with phosphate-ethylated and methyl phosphonate systems, phosphate-methylated systems are preferable as antisense DNA, which was concluded from the high Tm values and sharp melting transitions of duplexes of phosphate-methylated and natural DNA. By using the Sanger dideoxy technique, it was shown that a complementary phosphate-methylated 18-mer can effectively and site-specifically block the DNA replication process at room temperature.

Retracted HIV Study Provides New Information about the Status of the <i>in</i> <i>Vitro</i> Inhibition of DNA Replication by Backbone Methylation

“…The correctness of the synthetic part of the Technical Comment obtains a doubtful or negative assessment with the quote: "This result contrasts with earlier statements that the degree of phosphate-methylation of tested DNA was 90% to 100%" (see Regiospecific inhibition of DNA duplication by antisense phosphate-methylated oligonucleotides of Moody et al [7]). These experiments were carried out with long and short modified DNA's (18-and 8-mers), prepared according to Koole's procedure.…”

“…The procedure for relatively long fragments was developed by Koole et al [7] [9] and based on the following reaction sequence:…”

“…the regiospecific inhibition of DNA duplication and the 1H NMR data, there is a distinct indication that the phosphate-backbone in the natural DNA was methylated [7].…”

“…The Retraction mainly focused on the small degree of methylation of the 20-nucleotide DNA's. These results conflict with their achievement in the in vitro regiospecific inhibition of DNA duplication with long and short fragments, prepared in a similar manner as in the HIV-study [7]. The suppression of the DNA polymerase I (Klenow fragment) in the inhibition experiments with the neutrally backbone-modified DNA's as inhibitors, has been explicitly demonstrated and may be considered as an additional support for the synthesis of the methylated DNA backbones [7].…”

The Chemical and Biochemical Properties of Methylphosphotriester DNA

“…A control experiment in which the complementarity is absent showed no decrease of the relative synthesis activity along the template strand. [41]c b An 18-mer complexed in this way has been used to test its hybridization ability over its full length with tRNA Phe directed on the amino acid stem and TcC arm. However, the interpretation of the corresponding NMR spectrum given in Ref.…”