title: Conformationally restricted nucleotides as a probe of structure-function relationships in RNA.
abstract: We introduce the use of commercially available locked nucleic acids (LNAs) as a functional probe in RNA. LNA nucleotides contain a covalent linkage that restricts the pseudorotation phase of the ribose to C3'-endo (A-form). Introduction of an LNA at a single site thus allows the role of ribose structure and dynamics in RNA function to be assessed. We apply LNA probing at multiple sites to analyze self-cleavage in the lead-dependent ribozyme (leadzyme), thermodynamic stability in the UUCG tetraloop, and the kinetics of recognition of U1A protein by U1 snRNA hairpin II. In the leadzyme, locking a single guanosine residue into the C3'-endo pucker increases the catalytic rate by a factor of 20, despite the fact that X-ray crystallographic and NMR structures of the leadzyme ground state reported a C2'-endo conformation at this site. These results strongly suggest that a conformational change at this position is critical for catalytic function. Functional insights obtained in all three systems demonstrate the highly general applicability of LNA probing in analysis of the role of ribose orientation in RNA structure, dynamics, and function.
authors.affiliation: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824, USA.
authors.forename: Kristine R
authors.initials: KR
authors.lastname: Julien
authors.forename: Minako
authors.initials: M
authors.lastname: Sumita
authors.forename: Po-Han
authors.initials: PH
authors.lastname: Chen
authors.forename: Ite A
authors.initials: IA
authors.lastname: Laird-Offringa
authors.forename: Charles G
authors.initials: CG
authors.lastname: Hoogstraten
mesh_terms: D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000378:metabolism*; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000737:chemistry* / Q000378:metabolism*; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000378:metabolism; D012266:Ribose / Q000737:chemistry; D013329:Structure-Activity Relationship; D013816:Thermodynamics
chemical_list: D009841:Oligonucleotides; D016337:RNA, Catalytic; D012342:RNA, Small Nuclear; D017412:Ribonucleoprotein, U1 Small Nuclear; C405483:U1 small nuclear RNA; C075339:U1A protein; C477371:locked nucleic acid; D012266:Ribose