Template-mediated synthesis of lariat RNA and DNA.
Nucleic acid "lariats" have been of great interest to the biological community since their discovery two decades ago as splicing intermediates in the biosynthesis of messenger RNA (lariat RNA introns). We report here the first synthesis of lariat DNA and RNA via template-mediated chemical ligation of Y-shaped oligonucleotides. The method allows for the synthesis of lariat DNA of any base composition as well as the more biologically relevant lariat RNA. Typically, branched precursors and complementary linear templates ("splints") were dissolved in an equimolar ratio at a total concentration of 10(-4) M, and ligation was promoted by addition of cyanogen bromide in a pH 7.6 buffer. The template-directed cyclization was very efficient, since the amount of circularized lariat product observed in all cases was in the 40-60% range. The lariats were purified by polyacrylamide gel electrophoresis, and their structure and nucleotide composition confirmed by MALDI-TOF mass spectrometry. Thermal denaturation and circular dichroism studies of lariat:RNA and lariat:DNA duplexes were fully supportive of the isolated "lasso" structures. Further characterization was conducted by enzymatic degradation with spleen phosphodiesterase (a 3'-exonuclease) and the RNA lariat debranching enzyme, a specific 2',5'-phosphodiesterase.
Department of Chemistry, Otto Maass Chemistry Building, McGill University, 801 Sherbrooke Street West, Montreal, QC, H3A 2K6, Canada.
Sandra
S
Carriero
Masad J
MJ
Damha
D002942:Circular Dichroism; D003488:Cyanogen Bromide / Q000737:chemistry; D003500:Cyclization; D004247:DNA / Q000138:chemical synthesis* / Q000235:genetics; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D009691:Nucleic Acid Denaturation; D009843:Oligoribonucleotides / Q000138:chemical synthesis*; D010727:Phosphoric Diester Hydrolases; D012313:RNA / Q000138:chemical synthesis* / Q000235:genetics; D012316:RNA Nucleotidyltransferases / Q000737:chemistry*; D012322:RNA Precursors / Q000737:chemistry; D012333:RNA, Messenger / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature; D013698:Templates, Genetic
D009843:Oligoribonucleotides; D012322:RNA Precursors; D012333:RNA, Messenger; D012313:RNA; D004247:DNA; D012316:RNA Nucleotidyltransferases; C048135:lariat debranching enzyme; D010727:Phosphoric Diester Hydrolases; D003488:Cyanogen Bromide