| Abstract [eng] |
The aim of the dissertation was to create an effective and specific method for labeling of miRNA and siRNA duplexes, suitable for detection or purification of these molecules, using recombinant small RNA methyltransferase HEN1 from Arabidopsis thaliana and synthetic its cofactor S-adenosil-L-methionine (AdoMet) analogues. The natural mechanism of methylation of miRNA/miRNA* and siRNA/siRNA* duplexes was investigated and factors important to the reaction were determined. The methyltransferase was applied for transferring of side chains with functional or reporter groups from synthetic AdoMet cofactor analogues to miRNA/miRNA* and siRNA/siRNA* duplexes. Moreover, it was figured out that HEN1 is able to modify not only typical RNA/RNA duplexes, but also RNA strand pre-annealed with DNA, RNA-LNA (locked nucleic acid) or DNA-LNA, as well as blunt-ended or chemically-modified substrates. One and two-step labeling technologies were created based on this research, suitable for miRNA and siRNA labeling with biotin for its purification or for specific labeling with fluorophores, including Cy5, Cy5.5 and etherneon. Also, methods for miRNA and siRNA enrichment using streptavidin-specific aptamer and double-labeling method small RNA-specific FRET technology were developed. |
