M. N. Repkova

Positioning of the mRNA stop signal with respect to polypeptide chain release factors and ribosomal proteins in 80S ribosomes

To study positioning of the mRNA stop signal with respect to polypeptide chain release factors (RFs) and ribosomal components within human 80S ribosomes, photoreactive mRNA analogs were applied. Derivatives of the UUCUAAA heptaribonucleotide containing the UUC codon for Phe and the stop signal UAAA, which bore a perfluoroaryl azido group at either the fourth nucleotide or the 3′‐terminal phosphate, were synthesized. The UUC codon was directed to the ribosomal P site by the cognate tRNAPhe, targeting the UAA stop codon to the A site. Mild UV irradiation of the ternary complexes consisting of the 80S ribosome, the mRNA analog and tRNA resulted in tRNA‐dependent crosslinking of the mRNA analogs to the 40S ribosomal proteins and the 18S rRNA. mRNA analogs with the photoreactive group at the fourth uridine (the first base of the stop codon) crosslinked mainly to protein S15 (and much less to S2). For the 3′‐modified mRNA analog, the major crosslinking target was protein S2, while protein S15 was much less crosslinked. Crosslinking of eukaryotic (e) RF1 was entirely dependent on the presence of a stop signal in the mRNA analog. eRF3 in the presence of eRF1 did not crosslink, but decreased the yield of eRF1 crosslinking. We conclude that (i) proteins S15 and S2 of the 40S ribosomal subunit are located near the A site‐bound codon; (ii) eRF1 can induce spatial rearrangement of the 80S ribosome leading to movement of protein L4 of the 60S ribosomal subunit closer to the codon located at the A site; (iii) within the 80S ribosome, eRF3 in the presence of eRF1 does not contact the stop codon at the A site and is probably located mostly (if not entirely) on the 60S subunit.

Photomodification of RNA and DNA fragments by oligonucleotide reagents bearing arylazide groups

Photomodification of ribo- and deoxyribo-octanucleotides by oligonucleotide reagents (6- and 7-mers) bearing p-azidotetrafluorobenzamido and 2-nitro-5-azidobenzamido groups has been investigated. It is shown that the oligonucleotides with a perfluoroarylazide group were effective modifiers both of deoxyribo- and ribo-targets. Maximum extent of cross-linked product formation (70%) was obtained when the deoxyribo-octanucleotide was modified by a heptanucleotide reagent with a perfluoroarylazide group. Selectivity of the photomodification was also high (50% on the G-residue at a certain position).

Silencing of MDR 1 gene in cancer cells by siRNA

Inhibition of p‐glycoprotein (PGP) expression and reverse of multidrug resistance (MDR) phenotype in KB‐8‐5 cells by synthetic 21‐bp double‐stranded oligoribonucleotides were investigated. siRNA constructs for the efficient down regulation of MDR1 that are active in nanomolar concentrations and cause reversal of MDR phenotype in cells were developed.

The mRNA Codon Environment at the P and E Sites of Human Ribosomes Deduced from Photocrosslinking with pUUUGUU Derivatives

Three mRNA analogs—derivatives of hexaribonucleotide pUUUGUU comprising phenylalanine and valine codons with a perfluoroarylazido group attached to the C5 atom of the uridine residue at the first, second, or third position—were used for photocrosslinking with 80S ribosomes from human placenta. The mRNA analogs were positioned on the ribosome with tRNA recognizing these codons: UUU was at the P site if tRNAPhe was used, while tRNAVal was used to put there the GUU codon (UUU at the E site). Thus, the crosslinking group of mRNA analog might occupy positions –3 to +3 with respect to the first nucleotide of the codon at the P site. Irradiation of the complexes with mild UV light (λ > 280 nm) resulted in the crosslinking of pUUUGUU derivatives with 18S RNA and proteins in the ribosome small subunit. The crosslinking with rRNA was observed only in the presence of tRNA. The photoactivatable group in positions –1 to +3 binds to G1207, while that in positions –2 or –3 binds to G961 of 18S RNA. In all cases, we observed crosslinking with S2 and S3 proteins irrespective of the presence of tRNA in the complex. Crosslinking with S23 and S26 proteins was observed mainly in the presence of tRNA when modified nucleotide occupied the +1 position (for both proteins) or the –3 position (for S26 protein). The crosslinking with S5/S7 proteins was substantial when modified nucleotide was in the –3 position, this crosslinking was not observed in the absence of tRNA.

High-performance method for specific effect on nucleic acids in cells using TiO2~DNA nanocomposites

Nanoparticles are used to solve the current drug delivery problem. We present a high-performance method for efficient and selective action on nucleic acid target in cells using unique TiO2·PL-DNA nanocomposites (polylysine-containing DNA fragments noncovalently immobilized onto TiO2 nanoparticles capable of transferring DNA). These nanocomposites were used for inhibition of human influenza A (H3N2) virus replication in infected MDCK cells. They showed a low toxicity (TC50 ≈ 1800 μg/ml) and a high antiviral activity (>99.9% inhibition of the virus replication). The specificity factor (antisense effect) appeared to depend on the delivery system of DNA fragments. This factor for nanocomposites is ten-times higher than for DNA in the presence of lipofectamine. IC50 for nanocomposites was estimated to be 1.5 μg/ml (30 nM for DNA), so its selectivity index was calculated as ~1200. Thus, the proposed nanocomposites are prospective for therapeutic application.

Arrangement of mRNA 3' of the A site codon on the human 80S ribosome.

Positioning of mRNA 3’ of the A site codon was studied with the use of short mRNA analogues carrying a UUU triplet at the 5’-termini and a perfluorophenylazide group at either the N7 atom of the guanosine or the C5 atom of the uridine 3’ of the triplet. Modified nucleotides were directed to positions +7, +9 or +12 with respect to the first nucleotide of the P site codon by tRNAPhe cognate to the UUU triplet targeted to the P site. Mild UV-irradiation resulted in cross-linking of the mRNA analogues to the 18S rRNA and to 40S proteins, the yield of cross-linking depending on the nature of the mRNA nucleotide bearing the modified group and its position on the ribosome. In addition, the yield of cross-linking to the 18S rRNA decreased strongly when the modified nucleotide was moved from position +7 to position +12. All the mRNA analogues cross-linked to the 18S rRNA nucleotides that had been found earlier at the decoding site, namely, to the invariant dinucleotide A1824/A1825 and the variable A1823 in the 3’-minidomain of the 18S rRNA, and also to the invariant nucleotide C1698 in the 3’-minidomain and the conserved region 605-620 in the apical region of helix 18 in the 5’-domain. The results indicate that (i) the mRNA makes a sharp turn between positions +6 and +7, (ii) the triplet immediately 3’ of the A site codon neighbors the 18S rRNA and proteins, and (iii) the codon 3’ of the triplet mentioned is surrounded mainly by proteins.

Interaction of primer tRNALys3 with the p51 subunit of human immunodeficiency virus type 1 reverse transcriptase: a possible role in enzyme activation

In the interaction between HIV‐1 RT and tRNALys3 each subunit of the heterodimer interacts with tRNA showing a different affinity: K d (p66) = 23 nM, K d (p51) = 140 nM. Preincubation of heterodimeric RT with tRNA, at concentrations similar to that of the K d value for p51, leads to an increase of the catalytic activity on poly(A)‐oligo(dT). These results were compared to those using different tRNA analogs: oxidized tRNA, lacking one, two or three nucleotides from the 3′‐end, or ribo‐ and deoxyribonucleotides mimicking the anticodon loop sequence. In all cases, tRNA analogs were weaker activators of HIV‐1 RT than natural tRNA. A possible mechanism of RT p66/p51 activation by tRNA and its analogs, mediated through the p51 subunit, is discussed.

Catalytic DNA and RNA for Targeting MDR1 mRNA

Abstract Design, synthesis and properties of catalytic NAs for targeting MDR1 mRNA are reported.

Efficient inhibition of human influenza a virus by oligonucleotides electrostatically fixed on polylysine-containing TiO2 nanoparticles

Antiviral activity of the TiO2·PL·DNA nanobiocomposites was studied on the MDCK cell culture infected with influenza A virus (subtype H3N2). DNA fragments in the nanocomposites are electrostatically bound to titanium dioxide nanoparticles precovered with polylysine. It was shown that TiO2·PL·DNA(v3′) nanocomposite bearing the DNA(v3′) fragment targeted to the 3′-end of the noncoding region of segment 5 of viral RNA specifically inhibited the virus reproduction with the efficiency of 99.8% and 99.9% (i.e., by factors of ∼400 and 1000, respectively) at a low concentration of DNA(v3′) in nanocomposite (0.1 and 0.2 μM, respectively). The TiO2·PL·DNA(r) nanocomposite containing an oligonucleotide noncomplementary to viral RNA or oligonucleotide DNA(v3′) unbound to the nanoparticles show very low antiviral activity (inhibition by factors of ∼3.5 and 1.3, respectively).

Two‐Component 10–23 DNA Enzymes

A new strategy for engineering of catalytic two‐component constructions based on 10–23 DNAzyme was proposed. The using of a combination of shortened DNAzyme with 2′‐O‐methyl oligomers as effectors significantly increased the catalytic activity of this DNAzyme.