title: Synthesis of a biotinylated photocleavable nucleotide monophosphate for the preparation of natively folded RNAs.
abstract: RNAs are involved in many functional roles in the cell, and this functional diversity is predicated on RNAs adopting requisite three-dimensional architectures. Preparing such "natively folded" RNAs with a homogeneous population is sometimes problematic for structural or enzymatic studies. Yet, standard methods for RNA preparations denature the RNA and create a heterogeneous population of conformers. Therefore, preparation of "natively folded" RNAs without going through the process of denaturing and refolding is important to obtain maximal biological function. 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 is readily accepted by T7 RNA polymerase to transcribe "natively folded" RNAs ranging in size from 27 to 493 nucleotides. This facile strategy allows efficient biotinylation of RNA and provides a traceless means to remove the biotin after the purification. Such preparation of natively folded RNAs should benefit biophysical and therapeutic applications.
authors.affiliation: Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland, USA.
authors.forename: Yiling
authors.initials: Y
authors.lastname: Luo
authors.affiliation: Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland, USA. Electronic address: hsintim@umd.edu.
authors.forename: Herman O
authors.initials: HO
authors.lastname: Sintim
authors.affiliation: Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland, USA; Center for Biomolecular Structure and Organization, University of Maryland, College Park, Maryland, USA. Electronic address: dayie@umd.edu.
authors.forename: T Kwaku
authors.initials: TK
authors.lastname: Dayie
mesh_terms: D001710:Biotin / Q000737:chemistry; D019921:Biotinylation; D002846:Chromatography, Affinity / Q000379:methods; D057930:Click Chemistry / Q000379:methods*; D006157:Guanosine Monophosphate / Q000138:chemical synthesis / Q000737:chemistry*; D010782:Photolysis; D012313:RNA / Q000138:chemical synthesis / Q000737:chemistry* / Q000302:isolation & purification; D059370:RNA Folding
chemical_list: D012313:RNA; D001710:Biotin; D006157:Guanosine Monophosphate
keywords: Affinity purification; Click chemistry; Native preparation; Nucleotide synthesis; Photochemical release; Purification; RNA