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44424-AC4
Improving the Function of RNA by Conformational Restriction
During this period of funding, we have accomplished three major goals: 1.) Applying conformationally restricted nucleotides to dissecting and improving the function of a catalytic RNA, 2.) Assessing the thermodynamic basis for ligand specificity by an RNA aptamer, and 3.) Reviewed the physical chemistry of RNA hairpin folding, with a focus on conformational restriction of RNA by nucleotide analog incorporation.
1.) The lead-dependent ribozyme, or leadzyme, uses a lead ion as a co-factor to self-cleave. Three structural models for the leadzyme have been advanced. These come from NMR, crystallography, and computational studies. However, all three structures differ in essential ways at the active site. One key question has been, “Which, if any, of these structures of the leadzyme are functionally relevant?” Fortunately, each of the structures has a unique guanosine at the active site in the unusual syn conformation. We replaced each of the guanosines with 8-bromoguanosine (8BrG), which is biased into the syn conformation, singly and assayed the effect of the substitution on ribozyme activity. In the cases of the substitutions relevant to the NMR and X-ray structures, the changes led to substantially reduced enzymatic activity. However, in the case of the computational model, enzymatic activity was enhanced by up to 30-fold. These findings indicate that, in at least some instances, computational methods can provide more functionally relevant structures than experimental methods. This result probably occurs because the computational models are built up from constraints that originate from experiments such as kinetics and thermodynamics. Findings from this study suggest that conformationally restricted nucleotides might be useful in judging the functional relevance of other RNA molecules. In addition, judicious insertion of such nucleotides could bias RNA molecules to fold in more functionally relevant conformations for use in experimental structure determination methods of NMR and X-ray crystallography.
2.) We have compared the thermodynamic parameters for binding of cognate and non-cognate ligands to an RNA aptamer. In particular, malachite green (MG) and tetramethylrosamine (TMR) are cognate and non-cognate ligands, respectively, to the malachite green aptamer. Surprisingly, the non-cognate ligand has been reported to bind tighter than the cognate ligand. We have been investigating the thermodynamic basis for this reverse discrimination by isothermal titration calorimetry (ITC). Strikingly, the cognate ligand gives a more favorable enthalpic interaction term, as well as a two-fold larger heat capacity change upon ligand binding than the non-cognate ligand. The change in heat capacity is often associated with specificity in DNA-protein interactions, and thus appears to carry over to RNA-ligand interactions as well. These findings suggest that large-scale conformational changes may be characteristic of cognate interactions in RNA. Moreover, it is possible that substrate specificity could be reversed by rational insertion of conformationally restricted nucleotides, which could impact the entropy of binding in favorable ways.
3.) RNA hairpins are ubiquitous in biology. We wrote a review on the physical chemistry of RNA hairpins for Annual Reviews of Physical Chemistry, with an emphasis on the function of RNA hairpins in biology. One focus of the review was the role and application of conformationally restricted nucleotides in biology. Special attention was placed on identifying key unanswered questions in the field. It is hoped that this article will stimulate physical chemists to work on RNA hairpin folding with the hope that new advances in understanding how RNA folding thermodynamics and kinetics impact biological function will be attained.
The support of the ACS-PRF has impacted my career in many ways. It has provided the support to work on conformationally restricted nucleotides in RNA function and folding in ways that were not otherwise possible. This has allowed me to provide support to outstanding graduate and undergraduate students, and to provide materials for the experiments. One graduate student (Rieko Yajima) is now a Senior Program Associate at AAAS's Research Competitive program, while another student (Joshua Blose) is continuing on his way towards the Ph.D. An undergraduate (Sarah Krahe) has contributed substantially to the project. She is now a senior and will be writing her honors thesis on this project. She is planning to get an advanced degree in pharmacy.
