title: RNAs synthesized using photocleavable biotinylated nucleotides have dramatically improved catalytic efficiency.
abstract: Obtaining homogeneous population of natively folded RNAs is a crippling problem encountered when preparing RNAs for structural or enzymatic studies. Most of the traditional methods that are employed to prepare large quantities of RNAs involve procedures that partially denature the RNA. Here, we present a simple strategy using 'click' chemistry to couple biotin to a 'caged' photocleavable (PC) guanosine monophosphate (GMP) in high yield. This biotin-PC GMP, accepted by T7 RNA polymerase, has been used to transcribe RNAs ranging in size from 27 to 527 nt. Furthermore we show, using an in-gel fluorescence assay, that natively prepared 160 and 175 kDa minimal group II intron ribozymes have enhanced catalytic activity over the same RNAs, purified via denaturing conditions and refolded. We conclude that large complex RNAs prepared by non-denaturing means form a homogeneous population and are catalytically more active than those prepared by denaturing methods and subsequent refolding; this facile approach for native RNA preparation should benefit synthesis of RNAs for biophysical and therapeutic applications.
authors: Department of Chemistry and Biochemistry, Center for Biomolecular Structure & Organization, University of Maryland, 1115 Biomolecular Sciences Bldg, College Park, MD 20742-3360, USA.
authors: Yiling
authors: Y
authors: Luo
authors: Nadukkudy V
authors: NV
authors: Eldho
authors: Herman O
authors: HO
authors: Sintim
authors: T Kwaku
authors: TK
authors: Dayie
mesh_terms: D001360:Avidin / Q000737:chemistry; D001483:Base Sequence; D019921:Biotinylation; D002384:Catalysis; D002846:Chromatography, Affinity; D057930:Click Chemistry; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D006157:Guanosine Monophosphate / Q000737:chemistry*; D007438:Introns; D008969:Molecular Sequence Data; D055668:Photochemical Processes; D012313:RNA / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D016337:RNA, Catalytic / Q000096:biosynthesis* / Q000378:metabolism; D012335:RNA, Ribosomal / Q000378:metabolism; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000378:metabolism
chemical_list: D016337:RNA, Catalytic; D012335:RNA, Ribosomal; D014764:Viral Proteins; D001360:Avidin; D012313:RNA; D006157:Guanosine Monophosphate; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases