Part I Targeting RNA duplexes with modified triplex-forming oligonucleotides Part II Allostery within a Watson-Crick RNA duplex and its application in studying RNA recognition by RIG-I
Date of Issue2014
School of Physical and Mathematical Sciences
Part I: Targeting RNA Duplexes with Modified Triplex-Forming Oligonucleotides Triplex is emerging as an important RNA tertiary structure motif, in which consecutive base triples form between a duplex and a third strand. RNA duplex region is also often functionally important site for protein binding. Thus, Triplex-Forming Oligonucleotides (TFOs) may be developed to regulate various biological functions involving RNA such as viral ribosomal frame-shifting and reverse transcription. How chemical modification in TFOs affects RNA triplex stability, however, is not well understood. Here, we incorporated Locked Nucleic Acid (LNA), 2-thio U, and 2′-O methyl modified residues in a series of all pyrimidine RNA TFOs, and studied the binding to two RNA hairpin structures. 12-Base-triple major-groove pyrimidine•purine-pyrimidine triplex structures form between the RNA/DNA hairpins and the complementary RNA TFOs. UV-absorbance-detected thermal melting studies reveal that the LNA and 2-thio U modifications in TFOs strongly enhance triplex formation with both parental RNA and DNA duplex regions. In addition, we found that incorporation of 2′-O methyl modified residues in a TFO destabilizes and stabilizes triplex formation with RNA and DNA duplex regions, respectively. The (de)stabilization of RNA triplex formation may be facilitated through modulation of van der Waals contact, base stacking, hydrogen bonding, backbone preorganization, geometric compatibility, and/or dehydration energy. Better understanding of the molecular determinants of RNA triplex structure stability lays the foundation for designing and discovering novel sequence-specific duplex-binding ligands as diagnostic and therapeutic agents targeting RNA. Part II: Allostery within a Watson-Crick RNA Duplex and Its Application in Studying RNA Recognition by RIG-I RNA has rich local and global conformational dynamics, which may often result from allosteric effects. However, allostery within a Watson-Crick RNA duplex is not well known. Here, we report the direct observation that the local structural dynamics of a Watson-Crick RNA duplex is modulated allosterically by the specific binding of human Retinoic acid-inducible gene I (RIG-I) C-terminal domain (CTD). To detect the allosteric effect, we incorporated into Watson-Crick RNA duplexes a fluorescent analogue of adenine, 2-aminopurine (2AP), which is a sensitive probe of RNA local structure and dynamics. 2AP fluorescence increases upon ligand binding, suggesting ligand-induced changes of the local dynamics of the (2AP)-U pair. The enhancement of 2AP fluorescence is (1) distance dependent with the (2AP)-U pair positioned within about 13 base pairs away from the ligand binding site, and (2) structure dependent with no fluorescence changes observed upon the incorporation of a non-Watson-Crick pair in between the (2AP)-U pair and the ligand binding site. Binding of full-length RIG-I and a triplex-forming oligonucleotide (TFO) also induces 2AP fluorescence enhancement. Allosteric 2AP fluorescence changes facilitate quantifications of binding affinities. Thus, Watson-Crick RNA duplexes may not be considered as merely rigid structural scaffolds. Ligand binding- and tertiary interaction-induced allostery within Watson-Crick DNA/RNA duplexes may facilitate the regulation of functions involving nucleic acid structure dynamics and nucleic acid-protein assembly, such as catalysis, metabolite sensing, helicase/chaperone activity, and the detection of single mismatches and damaged/modified residues in nucleic acid duplexes.