The bacterial enzyme tRNA 2-selenouridine synthase (SelU) is responsible for the conversion of 5-substituted 2-thiouridine (R5S2U), present in the anticodon of some bacterial tRNAs, into 5-substituted 2-selenouridine (R5Se2U). We have already demonstrated using synthetic RNAs that transformation S2U→Se2U is a two-step process, in which the S2U-RNA is geranylated and the resulting geS2U-RNA is selenated. Currently, the question is how SelU recognizes its substrates and what the cellular pathway of R5S2U→R5Se2U conversion is in natural tRNA. In the study presented here, we characterized the SelU substrate requirements, identified SelU-associated tRNAs and their specific modifications in the wobble position. Finally, we explained the sequence of steps in the selenation of tRNA. The S2U position within the RNA chain, the flanking sequence of the modification, and the length of the RNA substrate, all have a key influence on the recognition by SelU. MST data on the affinity of SelU to individual RNAs confirmed the presumed process. SelU binds the R5S2U-tRNA and then catalyzes its geranylation to the R5geS2U-tRNA, which remains bound to the enzyme and is selenated in the next step of the transformation. Finally, the R5Se2U-tRNA leaves the enzyme and participates in the translation process. The enzyme does not directly catalyze the R5S2U-tRNA selenation and the R5geS2U-tRNA is the intermediate product in the linear sequence of reactions.

RNA interference (RNAi) technology provides a powerful molecular tool to reduce an expression of selected genes in eukaryotic cells. Short interfering RNAs (siRNAs) are the effector molecules that trigger RNAi. Here, we describe siRNAs that discriminate between the wild type and mutant (1174 C→G) alleles of human Presenilin1 gene (PSEN1). This mutation, resulting in L392V PSEN1 variant, contributes to early onset familial Alzheimer′s disease. Using the dual fluorescence assay, flow cytometry and fluorescent microscopy we identified positions 8th–11th, within the central part of the antisense strand, as the most sensitive to mismatches. 2‐Thiouridine chemical modification introduced at the 3′‐end of the antisense strand improved the allele discrimination, but wobble base pairing adjacent to the mutation site abolished the siRNA activity. Our data indicate that siRNAs can be designed to discriminate between the wild type and mutant alleles of genes that differ by just a single nucleotide.

Accumulation of insoluble aggregates of β-amyloid peptide, a cleavage product of amyloid precursor protein, is thought to be a central step in the pathogenesis of Alzheimer's disease. The major enzymes required for the generation of toxic amyloidbeta peptide are β- (BACE1) and γ-secretases. Here, we present the rational design and the application of synthetic and lentivirus vector-encoded siRNAs for specific and efficient knockdown of overexpressed and endogenous BACE1, both in dividing and neural stem cells and in a rat brain. We also tested an approach to anti-amyloid therapy by the use of the allele-specific siRNAs to silence the mutant presenilin 1 (L392V PS-1), the main component of γ-secretase, responsible for development of Familial Alzheimer's disease. Reducing the level of β-amyloid accumulation in the brain could be beneficial for metabolic studies as well as potential therapeutic approach for prevention and treatment of Alzheimer's disease.

S-Geranylated 2-thiouridines (geS2Us) are unique hydrophobic modified nucleosides identified very recently in bacterial tRNAs. Our study on the synthesis of geS2Ura-containing oligonucleotides (geS2U-RNA and geS2dU-DNA) revealed a fast substitution of the S-geranyl moiety by methylamine (frequently used in oligonucleotide deprotection procedures) or n-butylamine, providing the corresponding N2-alkyl isocytosine (R2isoCyt) derivatives. To retain the S-geranyl moiety in the DNA or RNA chains, the optimized deprotection protocol with 8 M ethanolic ammonia should be applied. The oligomers bearing the R2isoCyt heterocycle (R2isoC-RNA and R2isodC-DNA) are less hydrophobic than the corresponding S2U- and geS2U-modified oligomers, whereas, contrary to the previously reported data, geS2dU-DNA and geS2U-RNA exhibit significantly higher lipophilicity than the parent S2Ura-containing oligonucleotides. Thermodynamic studies revealed that: (a) both geS2Ura- and R2isoCyt-modified oligomers exhibit similar hybridization properties towards DNA and RNA templates, and (b) the R2isoCyt nucleobase preferentially hybridizes to guanine moiety in the DNA/DNA and RNA/RNA duplexes.