Application of directed evolution approaches to develop in vivo probes targeting modified RNAs

Recent research has brought ribonucleic acids (RNAs) to the forefront of understanding how cellular processes are controlled with the ever-expanding list of non-coding RNAs (ncRNAs)1â??4. In the vast majority of cases, ncRNAs enact their functions by binding with DNA, proteins, and other RNAs to effect a change3,5. These interactions can be very sensitive to small changes in sequences or chemical modifications. Indeed, post-transcriptionally modified RNAs have long been known to be of essential importance in a variety of well-studied cellular RNAs including both tRNAs and rRNAs. Furthermore, more recent studies with the widely distributed 6-methyladenine (m6A) base modification have shown their importance in the vast majority of cellular RNAs, including both coding and noncoding RNAs6.

Until recently, techniques for studying modified RNAs have largely been limited to mass spectrometry for identification of novel modifications as well as enzyme knockouts for studying the function of known RNA modifications. Recently, high-throughput techniques such as bisulfite sequencing7 and m6A-seq8 have advanced the field rapidly by allowing whole-genome profiling of RNA modification sites. However, these techniques analyze RNA samples after extraction outside of the in vivo environment and, as such, are susceptible to changing chemistry which can confound identification of modification types and locations9,10. Therefore, the ability to capture modified RNAs in the cellular context would reduce the likelihood of artificial chemical modifications by protecting RNAs during extraction while allowing similar sequencing approaches which are already in use for identification of novel modification sites.

In this work, we are taking advantage of directed evolution approaches to engineer natively expressing Escherichia coli proteins for high affinity and specificity against chemical modifications in RNAs. The scheme was tested using a well-studied protein, AlkB (alpha-ketoglutarate dependent dioxygenase), which naturally functions as a repair enzyme for m3C and m1A base methylations in DNA and RNA11,12. Initially, the display and selection system were validated using pre-defined pseudolibraries containing two previously characterized mutants of AlkB; alanine substitutions at amino acids 135 (D135A) and 69 (W69A) both destroy catalytic function while increasing or decreasing affinity for the modified RNA, respectively. Pools of these two displayed proteins in approximate ratios ranging from 1:100 to 1:10,000 were screened over nonspecifically methylated (methyl-methanesulfonate-treated) RNA targets to demonstrate enrichment of the higher affinity mutant D135A from a pool containing both mutants. Following validation of this approach, protein libraries were created from wild-type and mutant variants of AlkB using error-prone PCR. For directed evolution using full size libraries, synthetic 30-mer RNA containing m1A at a specified nucleotide was used as the target for screening. Our initial results show that specific affinity for binding modified RNAs can be engineered into native E. coli proteins.

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