Team:NTU-Singapore/Open Structured

Home Team Members Collaboration Attributions Description Mutation-internal Open / Structured Demonstrate Characterisation Improve Basic Parts Part Collection Human Practices Outreach Medal Criteria Judging Form

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Open/Structured RNA

Integrating our Human Practices

From our discussion with Dr. Wan Yue and Assoc Prof Xavier Roca, we were reminded that RNA tends to form secondary and tertiary structures in cells. As our dCasRx-ADAR2_DD editing is based on the formation of a duplex between the gRNA and the single-stranded target mRNA, they cautioned us that the presence of these structures in our target could inhibit gRNA binding, thereby preventing RNA editing. Thus, we sought to investigate whether RNA structure affects dCasRx-ADAR2_DD RNA editing by comparing editing rates in open (linear) and structured (tend to form secondary/tertiary structures) RNA. As we have yet to characterise our new parts at that juncture, we utilised dCasRx-ADAR2_DD (CasRx v1, or BBa_K2818001) from NTU-Singapore 2018 (which is also the background of our mutants) to investigate the RNA editing efficiency in these RNA. icSHAPE reactivity profiles and scores reports on RNA structure at a transcriptome-wide level. Low and high icSHAPE scores are indicative of secondary and linear structures respectively.^[1,2] Here, we used icSHAPE reactivity scores and RNA expression levels to predict candidate RNAs that form linear or secondary structures. From icSHAPE data, we selected 20 open and 20 structured candidate RNAs with high expression levels to ensure sufficient reads for targeted editing by dCasRx-ADAR2_DD.

Methodology

Results

Open RNA

Figure 1. Editing percentage on 20 open RNAs. (n=4)

Structured RNA

Figure 2. Editing percentage on 20 structured RNAs. (n=4)

Figure 3. Editing percentage of open and structured RNA on scatter plot. The middle black line refers to mean editing percentage. Flanking top and bottom lines are 1 standard deviation away from the mean.

Discussion and Conclusion

Given that structured RNA play critical roles in cellular regulation and function, dCasRx-ADAR2_DD-mediated RNA editing in structured RNA can have important implications for health and disease. Our results indicate that dCasRx-ADAR2_DD has no clear preference for open and structured RNA (Figure 3). This means that dCasRx-ADAR2_DD can target diverse RNA populations in the cell, making it useful for therapeutic and research applications. Another method that can more accurately predict RNA structures would be PARIS (psoralen analysis of RNA interactions and structures), which can resolve RNA structure at base pair level.^[3] In the future, PARIS information can be used to guide RNA structure prediction to further investigate the RNA editing activity of dCasRx-ADAR2_DD for open and structured RNA.

References

1. Flynn R, Zhang Q, Spitale R, Lee B, Mumbach M, Chang H. Transcriptome-wide interrogation of RNA secondary structure in living cells with icSHAPE. Nature Protocols. 2016;11(2):273-290.
2. Chan D, Feng C, Spitale R. Measuring RNA structure transcriptome-wide with icSHAPE. Methods. 2017;120:85-90.
3. Lu Z, Zhang Q, Lee B, Flynn R, Smith M, Robinson J et al. RNA Duplex Map in Living Cells Reveals Higher-Order Transcriptome Structure. Cell. 2016;165(5):1267-1279.