44424-AC4
Improving the Function of RNA by Conformational Restriction
During this period of funding, we pursued three aims: 1.) Development of a combinatorial method to incorporate conformationally restricted nucleotides (CRN) into RNA, 2.) Determination of the thermodynamic basis for ligand specificity in an RNA aptamer, and 3.) Investigation of the ability of an RNA with tertiary structure in Arabidopsis to be altered by ligand binding. 1.) In the previous report, we described using a guanosine analogue with a bias towards the syn conformation, 8-bromoguanosine (8BrG), to determine which of three structural models for the lead-dependent ribozyme (NMR, crystallography, or computational structure) was the most relevant to RNA catalysis. Surprisingly, these studies supported the computational structure as most relevant. This was rationalized in terms of the computational structure being derived from experimental constraints and the latter adopting non-reactive conformations. On the basis of this work, we suggested that CRNs might be useful in judging the functional relevance of other RNA molecules. Judicious insertion of such nucleotides could bias RNA molecules to fold into functionally relevant conformations for use in experimental structure determination by NMR and X-ray crystallography. Since that time, we have been testing ways to incorporate 8BrG and 8BrA into RNA using T7 transcription. We examined DNA templates of various structures, NTP mixtures, temperatures and reaction conditions. Overall, we find that we can readily incorporate 8BrA into a transcript. We can also incorporate 8BrGTP, albeit at a reduced efficiency. Efforts to improve 8BrGTP incorporation are currently underway and include doping, changing the start sequence, and using a mutant T7 RNA polymerase. Ultimately, we plan to incorporate 8BrGTP and/or 8BrATP at any given position in an RNA and screen for those that have enhanced function. 2.) We have furthered our studies of the thermodynamics 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 was found to bind tighter than the cognate ligand. We have been investigating the thermodynamic basis for this reverse discrimination by isothermal titration calorimetry (ITC). Last period, we reported that the cognate ligand gives a more favorable enthalpic interaction term, as well as a two-fold larger heat capacity change upon ligand binding. In the last year, we found that the cognate ligand binds with a stoichiometry near 0.4 at low temperature but a stoichiometry near 1 at higher temperature, while the non-cognate ligand binds with a stoichiometry near 1 at all temperatures. Thermal denaturation experiments on the aptamer both in presence and absence of MG and TMR ligands revealed that only the non-cognate TMR ligand has the ability to further stabilize the RNA and only in the presence of Mg2+, which drives tertiary structure. Together these results show that cognate and non-cognate ligands have the ability to alter the folding of the aptamer and the fraction that is in the native conformation. We are determining the nature of the active and inactive structures at low temperature. Ultimately, we are able to reconcile specificity of cognate ligand binding more with enthalpy than binding affinity, with cognate binding favored at the lowest temperatures tested. 3.) We have investigated the ability of certain mRNAs from the model plant Arabidopsis to acquire a tertiary structure and bind ligands. One long range goal is to conformationally restrict these RNAs through the use of CRNs and make sensors for ligand binding. Through comparative genomic techniques, we identified RNA elements that we believed to be riboswitches in the genome. These RNAs were prepared by molecular biological techniques and their folding studied by hydroxyl radical mapping and circular dichroism. Interestingly, we found that these RNAs acquire a tertiary structure in a Mg2+-dependent fashion. Unfortunately, efforts at identifying ligand binding by equilibrium dialysis have not been fruitful. Nonetheless, the presence of tertiary structures in these RNAs implies that they likely serve a functional role such as protein binding or protection of the RNA from reactive oxygen species during stress. These findings will be followed up in future studies. The support of the ACS-PRF has impacted my career in many ways. It has provided support to work on CRNs in RNA function and folding in ways that were not otherwise possible. This allowed me to provide support to outstanding graduate and undergraduate students, and to provide materials for 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 towards the Ph.D. An undergraduate (Sarah Krahe) has contributed substantially to the project. She is pursuing an advanced degree in pharmacy. Melissa Mullen is a graduate student investigating roles of RNA conformation in regulating gene expression in plants with an eye towards sensor development.
