Chimeric oligodeoxynucleotide analogs: Chemical synthesis, purification, and molecular and cellular biology protocols

Abstract

Publisher Summary The consequences of forming an intracellular antisense oligodeoxynucleotide (ON)-RNA heteroduplex may include inhibition of splicing, or other RNA-processing events, and inhibition of translation. Perhaps the most widely anticipated result is cleavage of the RNA, within the heteroduplex region, resulting from the action of the apparently ubiquitous endogenous enzyme ribonuclease H (RNase H). RNases H have been convincingly demonstrated to mediate many of the antisense effects observed in in vitro systems, following ON microinjection into Xenopus oocytes, eggs, and embryos, and following delivery of antisense effectors into human cells in culture. Clearly, if the transcript becomes degraded through the action of RNase H, then translation of the cognate protein is impossible. This chapter focuses on chimeric ON constructed with terminal methylphosphonodiester sections and a central phosphodiester region. Procedures for the synthesis, fluorescent labeling, purification, and use of chimeric ON are provided. All of the procedures described have been found to be robust and most are generally applicable to a wide range of antisense ON structures.