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Charakterystyka zjawiska interferencji RNA : podstawy strukturalne i właściwości siRNA

Sierant M., Nawrot B.

Wiadomości Chemiczne

2003

[Z] 57, 7-8

569--585

RNA interference (RNAi) is a natural biological mechanism for sequence-specific posttranscriptional gene silencing triggered by double-stranded RNA (dsRNA) homologous to a silenced gene. RNAi is found in a wide range of eukaryotes including human cells. The natural function of RNAi appears to be protection of a genome against invasion by mobile genetic elements such as transposons and viruses which produce aberrant RNA or dsRNA in a host cell. Specific mRNA degradation prevents transposon and virus replication. The majority of studies on the molecular mechanism underlying RNAi activity has been conducted in vivo using Drosophila melanogaster and Caenorhabditis elegans or in selected mammalian cell cultures. It has been demonstrated that long dsRNA is cleaved to 21–23 nucleotide long fragments by RNase III-like nuclease Dicer. These short interfering RNAs (siRNAs) are essential sequence–specific mediators of RNAi. They are bound by RNAi specific enzymes of nuclease complex RISC that targets mRNA for degradation. In this complex siRNA recognises, binds and cleaves the target mRNA. Cleavage occurs in the middle of the mRNA region recognized by the siRNA. The second model, which has been proposed for RNAi to explain the mechanism by which siRNA direct target mRNA destruction, requires RNA-dependent RNA polymerase (RdRP) to convert the target mRNA into dsRNA. RdRP is hypothesized to use antisense strand of siRNA as a primer in mRNA templated synthesis of complementary chain RNA. The resulting dsRNA is proposed to be cleaved then by Dicer for generation of secondary siRNA. Short interfering RNAs can be synthesized chemicaly or by in vitro transcription with T7 RNA polymerase, or expressed from siRNA coding vectors in the cells. These 21-nt siRNA duplexes cause efficent inhibition of exogenous and endogenous genes expression in a sequence-specific manner. Detailed analysis of potential modifications, that can be introduced into siRNA strands shows, that chemical modifications of sense strand are tolerated without loss of RNAi activity. However, some modification of antisense strand of siRNA (especially in the middle of the chain as well as modification of the 5’end) completely abolish RNAi. These results indicate that two strands of siRNA have different function in RNAi. RNAi approach can be broadly used for analysis of gene functions, and, what is even more important, this phenomenon can be used for searching new agents for therapeutic applications.

Selekcja kwasów nukleinowych in vitro

Nawrot B.

Wiadomości Chemiczne

2000

[Z] 54, 7-8

615-636

In vitro selection of nucleic acids (Systematic Evolution of Ligands by Exponential Enrichment - SELEX) is the selection technique which, from the library of randomised or degenerated RNA or DNA molecules, allows the isolation of nucleic acids with expected biochemical properties. Repeated cycles of selection and amplification have been used to isolate sequences of nucleic acids (aptamers) that bind with high affinity specific molecular targets - proteins or low molecular weight ligands. This method allows also to isolate a number of nucleic acids with catalytic activity and thus to generate new ribozymes and deoxyribozymes. SELEX is an useful tool of molecular biology and bioorganic chemistry for developing new therapeutic agents, as well for the insight in function of RNA in molecular evolution and RNA World hypothesis.