RNA molecules play important roles in all aspects of gene expression as well as many other cellular processes. Inside the cell, RNA molecules form distinct structures to interact with DNA, proteins, other RNAs and with various metabolites to carry out these functions. RNA molecules contain various recurrent structural features or motifs. This project aims to increase understanding of the details of bonding within recurrent RNA motifs and to elucidate rules for RNA structure formation. Colorado College undergraduates will measure the stabilities of small RNA structures using thermodynamic methods, including thermal denaturation and isothermal calorimetry. Particular regions of RNA will then be modified, and RNA stabilities before and after the modification will be compared to identify key contributors to bonding for each structural motif. Improved understanding of the general rules for structure formation will improve our ability to get greater information from the genome databases, to predict the structures formed by RNA in the cell and to elucidate their functions and mechanisms of action. Detailed thermodynamic parameters for recurrent RNA structures that guide and stabilize the folding of RNA as it is transcribed will improve our understanding of RNA folding, as these structures may serve as nucleation sites for the formation of higher-order structures.
Broader Impact: Broader impacts of this work include the scientific impact of improved knowledge of RNA structural motifs and recruitment and training of undergraduates in current research methods both inside and outside of the classroom. The laboratories in biochemistry courses are being modified to introduce research methodology to the students in the classroom. In nucleic acids biochemistry course, students will design an RNA-based project and apply their thermodynamic knowledge to understanding RNA structures. This project will strengthen their understanding of thermodynamics and RNA structure and function along introducing them to RNA research. Undergraduate students will be presented with an option to do research starting in their introductory classes. Up to eight undergraduate students will work in the research laboratory and participate in developing and testing hypotheses on small RNA structure and function. Women and minority students from biochemistry classes will be given priority to do research in the laboratory. In addition, undergraduate students will be provided opportunities to participate in outreach work with Longfellow and Audubon Elementary Schools. Elementary school students will learn the basic principles of science by testing hypothesis on concepts such as solubility and density. Students will make a positive contribution to the society by using their scientific knowledge to provide information about RNA to the general public on topics such as HIV's life cycle and its treatment; this in turn makes science relevant to their lives. The science outreach projects to the community are specifically designed to raise the scientific literacy of the general public. Sharing of equipment and expertise with University of Colorado, Colorado Springs will build bridges locally and will benefit all involved. High school students and teachers from Pine Creek High School will also participate in RNA research during the summer. Quantitative methods will be introduced into biology courses at Pine Creek High School.
Research in the last few decades has discovered a wide-range of new functions for RNA, from a role in catalysis, to gene silencing, to short RNA produced in response to viral infection in the bacterial cells. RNA is involved in multiple functions beyond its role in the production of proteins is evident by the fact that only ~ 5% of the human genome is expressed to code for proteins when nearly 75% of the genome is copied into RNA. Technologies utilizing small RNA, such as ribozymes, RNAi and CRISPR-Cas, are becoming significant for both basic and applied research. Thus, a better understanding of RNA structures and its functions is needed to understand, and perhaps manipulate, the cellular processes. RNA structures are made up of helical and non-helical regions. The non-helical or bulge regions make nearly 50% of the RNA and are formed by many non-canonical interactions. The research in our laboratory has focused on understanding the interactions that occur in small bulged RNA, especially those motifs that are implicated to be important for function of a larger RNA and are seen to bind to metal ions in crystal structures. We have examined small bulged RNA structures derived from RNA such as the thiamine pyrophosphate riboswitch, the TAR RNA of AIDS-causing human immunodeficiency virus, group I introns, etc. We have quantitated the effect of the bulge on RNA stability as a function of sequence and ion interactions. We find that the size of an asymmetric bulge has a significant effect on RNA stability and interactions with ions. RNA constructs are additionally stabilized by 1-4 kcal/mol in the presence of magnesium ions over one molar potassium ions depending on the sequence of the bulge. We are also examining the sequence context in which various non-canonical base pairs form. In the pyrophosphate sensor helix of TPP riboswitch two adjacent A+•C base pairs can form. All the research in the PI’s laboratory is done by undergraduate students. The students who work in the laboratory are typically given their own project for the duration of their time in the laboratory. These students are trained to read current scientific literature and are involved in designing their own experiments with the PI. Students trained in the laboratory primarily pursue graduate or medical schools. The students are also participating in organizations such as Teach for America, Peace Corp, Colorado Health Initiatives and are contributing their scientific knowledge to the benefit of the society. Students report research to be a significant contributor to their growth as a scientist and that it provides them with the independence and confidence needed to pursue the future scientific endeavors. Working on the timely (and expensive) biophysical experiments on RNA, only possible with the aid of the funding, is training students for technical work in the industry as they contemplate their future options. This funding has provided an opportunity to train 8-12 undergraduate students in research on RNA per year with sixteen additional students learning RNA and DNA techniques in the nucleic acid biochemistry class. Students are participating in outreach activities via the Bioscience Club and are bringing their scientific experience to the local elementary school. High school students and teachers also participated in the research during the summer. Thus, the funding has a large impact on number of students both inside and outside the classroom.
