LNA units present in the (2′-OMe)-RNA strand stabilize parallel duplexes (2′-OMe)-RNA/[All-R_P-PS]-DNA and parallel triplexes (2′-OMe)-RNA/[All-R_P-PS]-DNA/RNA. An improved tool for the inhibition of reverse transcription

Abstract

Homopurine phosphorothioate analogs of DNA, possessing all phosphorus atoms of RP configuration ([All-RP-PS]-DNA), when interact with appropriate complementary RNA or (2'-OMe)-RNA templates, form parallel triplexes or parallel duplexes of very high thermodynamic stability. The present results show that T-LNA or 5-Me-C-LNA units introduced into the parallel Hoogsteen-paired (2'-OMe)-RNA strands (up to four units in the oligomers of 9 or 12 nt in length) stabilize these parallel complexes. At neutral pH, dodecameric parallel duplexes have Tm values of 62-68 °C, which are by 4-10 °C higher than Tm for the reference duplex (with no LNA units present), while for the corresponding triplexes, Tm values exceeded 85 °C. For nonameric parallel duplexes, melting temperatures of 38-62 °C were found and (2'-OMe)-RNA oligomers containing 5-Me-C-LNA units stabilized the complexes more efficiently than the T-LNA containing congeners. In both series the stability of the parallel complexes increased with an increasing number of LNA units present. The same trend was observed in experiments of reverse transcription RNA→DNA (using AMV RT reverse transcriptase) where the formation of parallel triplexes (consisting of an RNA template, [All-RP-PS]-DNA nonamer and Hoogsteen-paired (2'-OMe)-RNA strands containing the LNA units) led to the efficient inhibition of the process. Under the best conditions checked (four 5-Me-C-LNA units, three-fold excess over the RNA template) the inhibition was 94% effective, compared to 71% inhibition observed in the reference system with the Hoogsteen-paired (2'-OMe)-RNA strand carrying no LNA units. This kind of complexation may "arrest" harmful RNA oligomers (e.g., viral RNA or mRNA of unwanted proteins) and, beneficially, exclude them from enzymatic processes, otherwise leading to viral or genetic diseases.

Structural Aspects of the Antiparallel and Parallel Duplexes Formed by DNA, 2’-O-Methyl RNA and RNA Oligonucleotides

“…Additionally, when LNA units were introduced in the 2’-O-Me RNA strand, the stabilizing effect was measurably stronger. These results are in accordance with data in the literature [ 35 , 38 , 39 ].…”

Parallel‐stranded DNA and RNA duplexes – structural features and potential applications

“…These results are supported by the findings of Kierzek's group . The same trend was observed in reverse transcription experiments, where the inhibition of the process by parallel triplexes with LNA moieties was more efficient than unmodified structures . Maciaszek et al .…”

“…In 2015, Guga's group reported that LNAs located in the 2′‐ O ‐methyl‐RNA strand enhanced the stability of parallel duplexes . The increase in thermodynamic stability of these structures correlates with the frequency of LNAs.…”

“…These results can be useful in the rational design of effective oligonucleotides that hybridize to a target in a parallel manner as well as the design of pH-sensitive molecular switches [29]. In 2015, Guga's group reported that LNAs located in the 2 0 -O-methyl-RNA strand enhanced the stability of parallel duplexes [32]. The increase in thermodynamic stability of these structures correlates with the frequency of LNAs.…”

Thermal stability and conformation of antiparallel duplexes formed by P-stereodefined phosphorothioate DNA/LNA chimeric oligomers with DNA and RNA matrices

“…3,4 It should be noted that a few years ago phosphorothioation of DNA in bacteria was discovered. 7,9,10 † Dedicated to Professor Andrzej Okruszek (Technical University of Lodz) on the occasion of his 70th birthday. 6 To achieve the desired stronger hybridization, one has to introduce more altering modifications into the sugar-phosphate backbone.…”