Synthesis of HIV-1 Ψ-site RNA sequences with site specific incorporation of the fluorescent base analog 2-aminopurine

摘要

Fluorescent nucleotide based analogs can serve as sensitive probes of the local structure and chemical environment of the base within a nucleic acid sequence. A significant strength of these base analogs is their similarity in molecular constitution and chemical properties to natural bases. While chemical synthesis has afforded the ability to generate oligonucleotides in good yield with sequence-specific incorporation of fluorescent based analogs, this method is limited in practice to the synthesis of relatively small RNAs of less than 80 nucleotides. Since most RNAs of biological interest are greater than ~80 nucleotides in length, methods for synthesizing these larger RNAs in good yield, while maintaining the ability to site-specifically incorporate base analogs that allow for fluorescence measurements, could be of broad interest. Here we describe an approach for synthesis of large RNA molecules (>100 nt) that uses T4 RNA ligase to segmentally join a sequence fragment of an RNA, chemically synthesized with a fluorescent based analog, with the remaining unmodified portion of the RNA oligonucleotide, synthesized through in vitro transcription with T7 polymerase. This method is demonstrated through synthesis of the packaging sequence (Ψ-site) derived from the HIV-1 genomic RNA leader sequence (~120 nt) with the fluorescent based analog, 2-aminopurine (2-AP), selectively incorporated into the dimerization initiation site (DIS) stem-loop. Using 2-AP fluorescence, RNA conformational changes associated with the formation of non-covalent DIS mediated Ψ-site dimers have been analyzed.