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Predictable control of RNA lifetime using engineered degradation-tuning RNAs

Abstract

The ability to tune RNA and gene expression dynamics is greatly needed for biotechnological applications. Native RNA stabilizers or engineered 5′ stability hairpins have been used to regulate transcript half-life to control recombinant protein expression. However, these methods have been mostly ad hoc and hence lack predictability and modularity. Here, we report a library of RNA modules called degradation-tuning RNAs (dtRNAs) that can increase or decrease transcript stability in vivo and in vitro. dtRNAs enable modulation of transcript stability over a 40-fold dynamic range in Escherichia coli with minimal influence on translation initiation. We harness dtRNAs in messenger RNAs and noncoding RNAs to tune gene circuit dynamics and enhance CRISPR interference in vivo. Use of stabilizing dtRNAs in cell-free transcription-translation reactions also tunes gene and RNA aptamer production. Finally, we combine dtRNAs with toehold switch sensors to enhance the performance of paper-based norovirus diagnostics, illustrating the potential of dtRNAs for biotechnological applications.

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Fig. 1: Modulation of RNA stability by native ompA stabilizer variants.
Fig. 2: Identifying functional structural features of synthetic dtRNAs.
Fig. 3: Using dtRNAs to modulate gene circuit dynamics and noncoding RNA levels.
Fig. 4: In vitro regulation of gene expression and RNA aptamer production via synthetic dtRNAs.
Fig. 5: Redesigned dtRNA/toehold switch sensors improve the performance of paper-based viral diagnostics.

Data availability

The data generated and/or analyzed during the current study are available from the corresponding authors on reasonable request. Source data are provided with this paper.

Code availability

The codes that support this study are available from the corresponding authors upon reasonable request.

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Acknowledgements

We thank Z. Yan for assistance with RNA preparation and in vitro gene expression experiments, and J. Cutts and N. Brookhouser for technical guidance for RT–qPCR experiments and data analysis. We also thank X. Tian, R. Zhang and Z. Mi for MATLAB assistance and useful discussions. This work was financially supported by a National Science Foundation grant (no. DMS-1100309) and National Institutes of Health (NIH) grant (nos. GM106081, GM131405) to X.W.; an NIH Director’s New Innovator Award (no. 1DP2GM126892), the Gates Foundation (grant no. OPP1160667), an Arizona Biomedical Research Commission New Investigator Award (no. ADHS16-162400), an Alfred P. Sloan Fellowship (no. FG-2017-9108), Gordon and Betty Moore Foundation funds (no. 6984), NIH funds (no. 1R21AI136571) and Arizona State University funds to A.A.G. The views, opinions and/or findings contained in this article are those of the authors and should not be interpreted as representing the official views or policies, either expressed or implied, of the NIH.

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Authors

Contributions

Q.Z. and X.W. conceived the research. Q.Z. and F.W. conducted in vivo gene expression regulation analysis by naturally occurring stabilizer variants and engineered the dtRNA library. X.C. and X.W. conducted mathematical modeling and data fitting analysis. Q.Z. performed dtRNA-regulated dynamic analysis. Q.Z. and K.S.-B. designed and tested the CRISPR interference system regulated by dtRNAs. Q.Z., D.M. and K.W. designed and performed the in vitro aptamer and GFP regulation assay. Q.Z., D.M. and K.W. redesigned the toehold sensors and performed in vitro norovirus diagnostics. Q.Z., D.M., K.S.-B., X.C., X.W. and A.A.G. analyzed the data. A.A.G and X.W. supervised the work. Q.Z., A.A.G. and X.W. wrote the paper.

Corresponding authors

Correspondence to Alexander A. Green or Xiao Wang.

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Peer review information Nature Chemical Biology thanks the anonymous reviewers for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Structure of ompA stabilizer and GFP expression measurement driven by a strong promoter.

a, Schematic showing the structure of naturally occurring ompA stabilizer. (b-c) GFP fluorescence measurement results for circuits driven a strong promoter. b, Design WT, Hp1 and Hp2 exhibits comparable GFP fluorescence. c, Each design with small structure formation nearby RBS region shows low GFP fluorescence levels. Data represent the mean ± s.d. of four biological replicates. P (WT_I) = 0.0083, P (Hp1_I) = 0.0084, and P (Hp2_I) = 0.0497. n.s. (not significant) P > 0.05, * P < 0.05, ** P < 0.01, P value is measured by two-tailed student’s t-test.

Source data

Extended Data Fig. 2 Fluorescence measurements on synthetic dtRNAs with inserted RNase E cleavage sites.

a, Fifteen synthetic dtRNAs are designed without (-) or with single/multiple RNase E cleavage sites (UCUUCC) engineered into different structural regions of the stable dtRNA. The inserted regions are marked yellow (right). Fluorescence measurement result shows that insertion of cleavage sites have minor effects on RNA stability. b, Fluorescence measurement for dtRNAs with multiple RNase E cleavage sites inserted into 18-nt loop region. c, Characterize the effect of dtRNA 5’ spacing length on GFP expression. Seven dtRNAs with 5’ spacing lengths from 1-nt to 18-nt are designed to measurement their effect on GFP expression. d, Fluorescence measurement of dtRNAs with RNase E cleavage sites engineered into 12-nt 5’ spacing region. All data represent the mean ± s.d. of six biological replicates.

Source data

Extended Data Fig. 3 Factors have minor effects on dtRNA function.

a, Relative GFP expression of circuits regulated by dtRNAs with or without the three-nucleotide bulge introduced in stem region. b, Fluorescence measurement result for designs with the same stem feature but varying loop GC content. c, Relative mRFP fluorescence regulated by selected dtRNAs with varying stabilizing abilities. Colors of the bar represent the fold enhancement of each dtRNA on GFP reporter. d, Comparison between relative mRFP fluorescence and relative GFP fluorescence regulated by selected dtRNAs. The result exhibits high correlation (R2 = 0.8681) between the report gene expression suggesting dtRNA performance is transferable to the other genes with different sequence composition. e, Commonality test for circuits with different promoters. Two promoters are selected (Biobrick number: J23105 and J23109, Supplementary Table 1) and engineered into the circuit with identical constructions. f, Commonality test for circuits with different RBSs. Two RBSs (Biobrick number: B0031 and B0032) are engineered into the circuit with identical constructions (Supplementary Table 1). All data represent the mean ± s.d. of six biological replicates.

Source data

Extended Data Fig. 4 qPCR measurement and dtRNAs function prediction.

a, RT–qPCR measurement of relative RNA levels for dtRNAs with diverse stabilizing efficiency. The result displays a strong correlation between relative RNA levels and relative GFP fluorescence (R2 = 0.9406). Data represent the mean ± s.d. of at least three biological replicates. b, Relative fluorescence comparison between predicted relative GFP and observed relative GFP of circuits constructed followed by combined design rules (Supplementary Table 3). N is the total number for 54 single measurement regulated by additional designed dtRNAs (R2 = 0.5005). c, Fluorescence measurement of dtRNA design f (Supplementary Table 3) without (left) or with (right) 18 nt 5’ spacing. Data represent the mean ± s.d. of six biological replicates. d, Scatter plot reveals that structure MFE is not significantly correlated with GFP fluorescence enhancement regulated by synthetic dtRNA library (R2 = 0.000068). Data represent the mean ± s.d. of six biological replicates.

Source data

Extended Data Fig. 5 Hysteresis measurement for dtRNA-regulated positive feedback loop.

a, Schematic showing the construction of positive feedback loop, dtRNA is only inserted at 5’ upstream of the LuxR gene. All genetic components are sharing the same colors as showed in Fig. 3a. b, The hysteresis result of Fig. 3c regulated by dR1 and dR82 induced by 0 to 2 nM 3OC6HSL concentration. This figure serves to zoom in on lower induction doses shown in Fig. 3c to better visualize low dosage dynamics. c, Hysteresis results for synthetic positive feedback circuit regulated by dR6 and dR81. Various concentrations of 3OC6HSL are applied to induce the circuit. The top panel is the enlarged result induced by 0 to 2 nM 3OC6HSL concentration. All data in b-c represent the mean ± s.d. of three biological replicates.

Source data

Extended Data Fig. 6 In vitro regulation of gene expression via synthetic dtRNAs.

a, GFP fluorescence measurement results of designs without RNase inhibitor treatment. b, GFP fluorescence measurement results of designs with RNase inhibitor treatment. All data represent the mean ± s.d. of three biological replicates. GFP fluorescence is measured every 50 seconds.

Extended Data Fig. 7 Relative GFP fluorescence comparison and in vitro dtRNA-regulated aptamer assay.

a, Relative GFP fluorescence comparison among circuits regulated by the same dtRNAs in vitro and in vivo. Data represent the mean ± s.d. of at least three biological replicates. b, Aptamer fluorescence measurement assay. c, Comparison between in vivo relative GFP fluorescence and relative aptamer fluorescence in cell-free expression system. The result shows little correlation between relative GFP and aptamer fluorescence. Interestingly, dtRNAs with short stem-loop hairpins tend to exert stronger positive effect on aptamer fluorescence (green dots). d, Aptamer fluorescence measurements with varying 5’ single-stranded length.

Source data

Extended Data Fig. 8 Two-hour in vitro norovirus diagnostics and the toehold sensor expression leakage.

a, Leaky expression of sensors Ori, dR19_1 dR19_4 and dR19_5 without RNase inhibitor treatment. Leaky expression indicates the false positive result that reporter expresses even without viral input. b, Plate reader measurement shows two-hour viral diagnostic result without RNase inhibitor treatment. ‘+’ represents groups induced by synthetic norovirus RNA and ‘–’ represents the negative control; The dash line indicates the detection threshold (ΔOD575 = 0.4). Data represent the mean ± s.d. of five biological replicates. c, Plate reader measurement shows device dR19_2 and dR19_3 exhibit high expression leakage. Data represents the mean ± s.d. of five biological replicates. d, Expression leakage of sensors Ori, dR19_1 dR19_4 and dR19_5 with RNase inhibitor treatment.

Source data

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Zhang, Q., Ma, D., Wu, F. et al. Predictable control of RNA lifetime using engineered degradation-tuning RNAs. Nat Chem Biol 17, 828–836 (2021). https://doi.org/10.1038/s41589-021-00816-4

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