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The Armitage Group |
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PNA Hybridization Probes for Biological Structure and Function |
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A central tenet of biological chemistry is that the function of a biological macromolecule (e.g. protein or nucleic acid) is strongly influenced by its three-dimensional structure. For example, unfolding of a protein enzyme usually results in complete loss of catalytic activity, even though the sequence of amino acids in the protein is unchanged. Similarly, formation of non-double-helical structures in DNA can cause up- or downregulation of gene expression. Naturally, developing a clear picture of how a biological process works requires fundamental knowledge about the structure as well as the function of the molecular components involved the pathway or reaction. The growing understanding of the central role played by RNA in the expression of genetic information is motivating intense research into RNA chemistry and biology. RNA not only serves as an intermediate between DNA and protein, it also catalyzes essential chemical reactions, including peptide bond formation in the ribosome, and directly regulates translation of messenger RNA in the form of siRNA, miRNA and other short RNA variants. Given our capabilities as well as the great interest in RNA in the Chemistry and Biological Sciences Departments at Carnegie Mellon, an important goal of our research group is to provide tools that can be used to probe the structure and/or function of RNA. High-resolution structural information can be obtained from NMR or X-ray crystallography, although these methods are difficult to use for the large RNAs usually found in biological systems. Fluorescence methods often provide lower-resolution, but still important information. A particularly useful method exploits Förster resonance energy transfer (FRET), wherein an excited dye molecule transfers it’s energy to a different, lower energy dye. This results in quenching of the fluorescence from the first (donor) dye and stimulation of fluorescence from the second (acceptor) dye, observed at longer wavelength. FRET is typically observed if the donor and acceptor are separated by less than 10 nm and thus, it can be used to monitor processes that result in changes in the donor-acceptor separation distance. An interesting application of FRET is to monitor the process of RNA splicing, which occurs in the nucleus of the cell. In humans (and many other organisms), the sequence of a gene usually codes for a protein that would be much longer than what is actually found when the protein is sequenced. The loss of information between DNA and protein occurs at the RNA level by a process known as splicing. During this process, RNA introns are excised out of the initially transcribed RNA and the remaining exons are stitched together to form the mature mRNA, which is subsequently translated into protein by the ribosome (Figure 1). Splicing is catalyzed by a large RNA-protein complex known as the spliceosome and, while much is known about the composition of the spliceosome, far less is known about the individual steps that occur during splicing. These steps include binding, conformational changes, dissociation and chemical reactions. We can begin to understand the overall mechanism of splicing by mapping out the structural changes that take place during the splicing process. In principle, FRET can be used to follow structural changes since the distance between appropriately placed donor and acceptor dyes should change in response to a conformational or chemical reaction step. However, RNA is not inherently fluorescent, so donor and acceptor dyes need to be introduced into the RNA structure. We use PNA to accomplish this by designing the PNA to be complementary to a specific site in the RNA. In addition, we synthesize the PNA bearing a fluorescent donor or acceptor dye. Mixing the PNA with the RNA allows the PNA to bind to its target site in the RNA, delivering the fluorescent dye to that specific location. Figure 1 illustrates how this strategy is used to follow splicing. Before splicing occurs, the donor and acceptor fluorophores are far apart due to the presence of the intron. However, after splicing, the donor-acceptor distance is much smaller and FRET can occur. We detect this as a decrease in donor fluorescence and an increase in acceptor fluorescence.
Figure 1. Schematic of RNA splicing reaction as followed by fluorescent PNA hybridization probes.
Collaborators. This project benefits from collaboration with Prof. Javier Lopez of the Biological Sciences department and Prof. Linda Peteanu of the Chemistry department. Prof. Lopez is an expert on RNA splicing while Prof. Peteanu is able to not only monitor fluorescence changes in real time but also at the single molecule level. Briefly, the Lopez lab provides the unspliced RNA as well as extract from cells that contain the components needed to assemble the spliceosome. Our lab provides the fluorescent PNA probes. The Peteanu lab immobilizes the fluorescent PNA-RNA complex onto a glass slide and then adds cell extract along with ATP to begin the splicing reaction. A fluorescence microscope is used to monitor the ratio of acceptor:donor fluorescence, which grows as splicing proceeds. Once the FRET ratio no longer changes, the reaction is assumed to be done. Then, the spliced RNA can be removed from the microscope and sequenced to verify that splicing actually occurred.
Future Work. In addition to delivering fluorescent dyes to specific locations in RNA, the PNA can introduce other types of probes or reagents. For example, a photocrosslinking agent could be attached to the PNA and used to covalently trap whatever is in the vicinity of the PNA binding site, which could be useful for analyzing structure of RNA-protein complexes. Alternatively, an RNA-cleavage agent could be delivered by the PNA, thus yielding an artificial ribonuclease. These projects will benefit from collaboration with Prof. Danith Ly of the Chemistry department. The synthetic modifications Prof. Ly’s group is making to the PNA leads to strongly enhanced binding affinity for RNA, meaning shorter PNAs can be used to bind to the RNA. This decreases the likelihood that the PNA will disrupt the inherent structure (and therefore the function!) of the RNA.
Personnel Kelly Robertson (5th year graduate student) Kim Zanotti (1st year graduate student)
Publications
1. Robertson, K. L.; Yu, L.; Armitage, B. A.; Lopez, A. J.; Peteanu, L. A. “Fluorescent PNA Probes as Hybridization Labels for Biological RNA” Biochemistry 2006, 45, 6066-6074.
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