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 C39-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 C39-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 C29-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.

Structure, dynamics, and mechanism of the lead-dependent ribozyme

“…Recently, a high-resolution ion-exchange HPLC-based assay for monitoring the full kinetic progress of leadzyme self-cleavage was also developed to resolve the 29,39-cyclic phosphate product from the first step and the 39-mono phosphate product from the second step in the early time traces (Figure 2B), as well as to monitor the conversion from the former to the latter (30). The wild type leadzyme cleaves with an overall kinetic rate constant of ;1.4 min -1 (t ;0.7 min) at 200 mM Pb 2q , consistent with earlier reports under similar conditions (25,26,29,33,38). The kinetics of two-step cleavage reaction can be described by the following four equations:…”

“…In the presence of Pb 2q , the leadzyme motif undergoes autocleavage of the phosphodiester bond between C6 and G7 in the longer strand. Subsequent structural and mechanistic studies often employed leadzyme constructs either as a unimolecular hairpin design capped by a stable GAAA tetraloop (25)(26)(27)(28)(29)(30) or in a two-piece duplex form (25,(31)(32)(33)(34)(35)(36)(37)(38).…”

“…Two recent exciting studies looked into whether locking either the base or sugar in a particular conformation site-specifically may favor or inhibit catalysis (29,38).…”

C2′-endo nucleotides as molecular timers suggested by the folding of an RNA domain

“…Examples include small RNA motifs, the spliceosome, and the ribosome (13,32,33). The A130 nucleotide in RNase P is typical in this respect because its interaction with A230 creates the local structure necessary for recognizing and binding the pre-tRNA substrate (Fig.…”

“…Most nucleotides in an RNA molecule exist in the C3Ј-endo conformation. Although less frequent, the C2Ј-endo conformation is highly overrepresented in catalytic active sites and in critical tertiary structures (12,13). At the static structure level, the C2Ј-endo conformation induces greater helical twist in an A-form helix (14,15) and spans a much longer 5Ј-to-3Ј distance (12,16).…”