Abstract:

Branched RNA molecules with a 2‘,5‘-phosphodiester linkage are important biochemical intermediates. Lariat RNA is a particular type of branched RNA that is formed during intron splicing in vivo. Synthesis of branched and lariat RNA is challenging, and there are few general approaches that are applicable in vitro. Here we report the identification of divalent metal-dependent deoxyribozymes (DNA enzymes) that synthesize branched and lariat RNA. In vitro selection was used to obtain deoxyribozymes that selectively join an internal RNA 2‘-hydroxyl with a 5‘-terminal triphosphate in a convenient “binding arms” format. At least 85% yield of 2‘,5‘-branched RNA is obtained at 37 °C and 20 mM Mn2+, pH 7.5 in ≤30 min, and for some DNA enzymes in as little as 2 min (kobs ≈ 0.1−2 min-1). This represents a rate enhancement of up to 5 million-fold over the background reaction. Lariat RNA is also synthesized by the new deoxyribozymes, which have significant potential as generalizable reagents for the practical preparation of branched and lariat RNA. Because nucleic acid enzymes apparently create branched RNA in nature (e.g., group II introns and the spliceosome), the new deoxyribozymes are of substantial mechanistic interest as well as practical importance.