Crystal structures of an A-form duplex with single-adenosine bulges and a conformational basis for site-specific RNA self-cleavage

AbstractBackground: Bulged nucleotides are common secondary structural motifs in RNA molecules and are often involved in RNA-RNA and RNA-protein interactions. RNA is selectively cleaved at bulge sites (when compared to other sites within stems) in the presence of divalent metal cations. The effects of bulge nucleotides on duplex stability and topology have been extensively investigated, but no detailed X-ray structures of bulge-containing RNA fragments have been available.Results: We have crystallized a self-complementary RNA-DNA chimeric 11-nucleotide sequence containing single-adenosine bulges under two different conditions, giving two distinct crystal forms. In both lattices the adenosines are looped out, leaving the stacking interactions in the duplex virtually unaffected. The bulges cause the duplex to kink in both cases. In one of the structures, the conformation of the bulged nucleotide places its modeled 2′-oxygen in line with the adjacent phosphate on the 3′ side, where it is poised for nucleophilic attack.Conclusions: Single adenosine bulges cause a marked opening of the normally narrow RNA major groove in both crystal structures, rendering the bases more accessible to interacting molecules compared with an intact stem. The geometries around the looped-out adenosines are different in the two crystal forms, indicating that bulges can confer considerable local plasticity on the usually rigid RNA double helix. The results provide a conformational basis for the preferential, metal-assisted self-cleavage of RNA at bulged sites

Nucleozymes

Nucleozymes containing ribonucleotides and deoxyribonucleotides or nucleic acid analogues are described herein. The nucleozymes have catalytic activity and are significantly more resistant to degradation than their all-RNA ribozyme counterparts. Also described are methods for preparing the nucleozymes along with methods of using nucleozymes, e.g., as therapeutic agents

Template-Directed Ligation of Peptides to Oligonucleotides

Synthetic oligonucleotides and peptides have enjoyed a wide range of applications in both biology and chemistry. As a consequence, oligonucleotide-peptide conjugates have received considerable attention, most notably in the development of antisense constructs with improved pharmacological properties. In addition, oligonucleotide-peptide conjugates have been used as molecular tags, in the assembly of supramolecular arrays and in the construction of encoded combinatorial libraries. To make these chimeric molecules more accessible for a broad range of investigations, we sought to develop a facile method for joining fully deprotected oligonucleotides and peptides through a stable amide bond linkage. Furthermore, we wished to make this ligation reaction addressable, enabling one to direct the ligation of specific oligonucleotide and peptide components.To confer specificity and accelerate the rate of the reaction, the ligation process was designed to be dependent on the presence of a complementary oligonucleotide template

Quantitating tertiary binding energies of 2′OH groups on the P1 duplex of the Tetrahymena ribozyme: Intrinsic binding energy in an RNA enzyme

ABSTRACT: Binding of the Tetrahymena ribozyme’s oligonucleotide substrate (S) involves P1 duplex formation with the ribozyme’s internal guide sequence (IGS) to give an open complex, followed by docking of the P1 duplex into the catalytic core via tertiary interactions to give a closed complex. The overall binding energies provided by 2 ′ OH groups on S and IGS have been measured previously. To obtain the energetic contribution of each of these 2 ′ OH groups in the docking step, we have separately measured their contribution to the stability of a model P1 duplex using “substrate inhibition”. This new approach allows measurement of duplex stabilities under conditions identical to those used for ribozyme binding measurements. The tertiary binding energies from the individual 2 ′ OH groups include a small destabilizing contribution of 0.7 kcal/mol and stabilizing contributions of up to-2.9 kcal/mol. The energetic contributions of specific 2 ′ OH groups are discussed in the context of considerable previous work that has characterized the tertiary interactions of the P1 duplex. A “threshold ” model for the open and closed complexes is presented that provides a framework to interpret the energetic effects of functional group substitutions on the P1 duplex. The sum of the tertiary stabilization provided by the conserved G‚U wobble at the cleavage site and the individual 2 ′ OH groups on the P1 duplex is significantly greater than the observed tertiary stabilization of S (11.0 vs 2.2 kcal/mol). It is suggested that there is an energeti