Thursday, April 3, 2025
4/3/2025
Accelerated evolution of biological RNAs - Project Summary: RNA is capable of diverse biological functions based on its ability to adopt well-defined structures. RNA also can serve as a genetic material that can be synthesized and amplified by protein polymerase enzymes. The application of selective pressures to an evolving population of RNAs in the laboratory enables the discovery of functional RNAs with user-specified properties. This process, the directed evolution of RNA, has been widely adopted to generate RNA-based diagnostics, therapeutics, cellular regulatory elements, and bio-imaging tools. Such synthetic functional RNAs are now instrumental to many research programs in biology and biomedicine, but the current paradigm for their development is inefficient and limits their utility. The protein polymerases that are used to evolve RNAs are not sufficiently error-prone to drive the rapid evolution of functional motifs that typically contain 20–50 nucleotides. The proposed research will develop an error-prone and sequence- unbiased RNA enzyme with robust RNA polymerase activity, tailored to accelerate the directed evolution of functional RNAs. In Aim 1, an error-prone polymerase ribozyme that is capable of synthesizing diverse functional RNAs will be adapted to the scaled production of mutagenized populations of RNAs for use in directed evolution campaigns. To achieve this goal, a novel microfluidics-based RNA evolution platform has been devised and its application to the current form of the RNA polymerase ribozyme has been validated. Simultaneously, in Aim 2, the conditions of the polymerization reaction will be optimized to achieve the desired error rates in the range of 2–5% per nucleotide position, with a broad and unbiased spectrum of mutations, thus enabling the productive mutagenesis of functional RNA motifs. The tools for accelerated RNA evolution developed in Aims 1 and 2 will then be applied in Aim 3 to generate new and improved forms of the Mango and coagulation factor IXa aptamers, which have utility in cellular imaging and anticoagulation, respectively. The accelerated RNA evolution platform developed here will broadly enhance the ability to generate bio-interfacing molecular probes and aptamers. The Joyce laboratory has pioneered the directed evolution of RNA. The laboratory’s expertise will be instrumental in the successful completion of the proposed evolution campaigns and in fostering the adoption of the accelerated RNA evolution platform by the broader scientific community. The Salk Institute houses state-of-the-art scientific core facilities that will support the FACS-based selection of RNA, deep sequencing and bioinformatics analyses of the evolving populations, and cellular imaging of the improved fluorescent aptamers. The Salk Institute and the broader La Jolla research community have remarkable and diverse capabilities in basic and applied biomedical research, which will provide the PI with a highly nurturing environment toward becoming a successful independent investigator.
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