| Abstract
| - Nucleic acid “lariats” have been of great interest to the biological community since their discoverytwo decades ago as splicing intermediates in the biosynthesis of messenger RNA (lariat RNAintrons). We report here the first synthesis of lariat DNA and RNA via template-mediated chemicalligation of Y-shaped oligonucleotides. The method allows for the synthesis of lariat DNA of anybase composition as well as the more biologically relevant lariat RNA. Typically, branched precursorsand complementary linear templates (“splints”) were dissolved in an equimolar ratio at a totalconcentration of 10-4 M, and ligation was promoted by addition of cyanogen bromide in a pH 7.6buffer. The template-directed cyclization was very efficient, since the amount of circularized lariatproduct observed in all cases was in the 40−60% range. The lariats were purified by polyacrylamidegel electrophoresis, and their structure and nucleotide composition confirmed by MALDI-TOF massspectrometry. Thermal denaturation and circular dichroism studies of lariat:RNA and lariat:DNAduplexes were fully supportive of the isolated “lasso” structures. Further characterization wasconducted by enzymatic degradation with spleen phosphodiesterase (a 3‘-exonuclease) and the RNAlariat debranching enzyme, a specific 2‘,5‘-phosphodiesterase.
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