1.
Hossain, G. S., Saini, M., Miyake, R., Ling, H. & Chang, M. W. Genetic
biosensor design for natural product biosynthesis in microorganisms. Trends Biotechnol. 38, 797–810 (2020).
2. Alexandrov, K. & Vickers, C. E. In vivo protein-based biosensors:
seeing metabolism in real time. Trends Biotechnol. 41, 19–26 (2023).
3. Carthew, R. W. Gene regulation and cellular metabolism: an
essential partnership. Trends Genet. 37, 389–400 (2021).
4. Chathuranga, K., Weerawardhana, A., Dodantenna, N. & Lee, J.-S.
Regulation of antiviral innate immune signaling and viral evasion
following viral genome sensing. Exp. Mol. Med. 53,
1647–1668 (2021).
5. Motwani, M., Pesiridis, S. & Fitzgerald, K. A. DNA sensing by the
cGAS-STING pathway in health and disease. Nat. Rev. Genet. 20,
657–674 (2019).
6. Gossen, M. et al. Transcriptional activation by tetracyclines in
mammalian cells. Science 268, 1766–1769 (1995).
7. Kavita, K. & Breaker, R. R. Discovering riboswitches: the past and the
future. Trends Biochem. Sci. 48, 119–141 (2023).
8. Raman, S., Taylor, N., Genuth, N., Fields, S. & Church, G. M. Engineering allostery. Trends Genet. 30, 521–528 (2014).
9. Jung, S. & Plückthun, A. Improving in vivo folding and stability of a
single-chain Fv antibody fragment by loop grafting. Protein Eng. 10,
959–966 (1997).
10. Wörn, A. et al. Correlation between in vitro stability and in vivo
performance of anti-GCN4 intrabodies as cytoplasmic inhibitors*. J.
Biol. Chem. 275, 2795–2803 (2000).
11. Gąciarz, A. & Ruddock, L. W. Complementarity determining regions
and frameworks contribute to the disulfide bond independent
folding of intrinsically stable scFv. PLoS ONE 12, e0189964 (2017).
12. Cao, J. et al. Nanobody-based sandwich reporter system for living
cell sensing influenza A virus infection. Sci. Rep. 9, 15899 (2019).
13. Cella, F., Wroblewska, L., Weiss, R. & Siciliano, V. Engineering
protein-protein devices for multilayered regulation of mRNA
translation using orthogonal proteases in mammalian cells. Nat.
Commun. 9, 4392 (2018).
Article
14. Nakanishi, H., Saito, H. & Itaka, K. Versatile design of intracellular
protein-responsive translational regulation system for synthetic
mRNA. ACS Synth. Biol. 11, 1077–1085 (2022).
15. Wang, W. et al. Bacteriophage T7 transcription system: an enabling
tool in synthetic biology. Biotechnol. Adv. 36, 2129–2137 (2018).
16. Ikeda, R. A. & Richardson, C. C. Enzymatic properties of a proteolytically nicked RNA polymerase of bacteriophage T7. J. Biol. Chem.
262, 3790–3799 (1987).
17. Shis, D. L. & Bennett, M. R. Library of synthetic transcriptional AND
gates built with split T7 RNA polymerase mutants. Proc. Natl. Acad.
Sci. USA 110, 5028–5033 (2013).
18. Pu, J., Zinkus-Boltz, J. & Dickinson, B. C. Evolution of a split RNA
polymerase as a versatile biosensor platform. Nat. Chem. Biol. 13,
432–438 (2017).
19. Pu, J., Kentala, K. & Dickinson, B. C. Multidimensional control of
Cas9 by evolved RNA polymerase-based biosensors. ACS Chem.
Biol. 13, 431–437 (2018).
20. Ueda, H. et al. Open sandwich ELISA: a novel immunoassay based
on the interchain interaction of antibody variable region. Nat. Biotechnol. 14, 1714–1718 (1996).
21. Hanes, J., Jermutus, L., Weber-Bornhauser, S., Bosshard, H. R. &
Plückthun, A. Ribosome display efficiently selects and evolves highaffinity antibodies in vitro from immune libraries. Proc. Natl. Acad.
Sci. USA 95, 14130–14135 (1998).
22. der Maur, A. A. et al. Direct in vivo screening of intrabody libraries
constructed on a highly stable single-chain framework. J. Biol.
Chem. 277, 45075–45085 (2002).
23. Steitz, T. A. The structural changes of T7 RNA polymerase from
transcription initiation to elongation. Curr. Opin. Struct. Biol. 19,
683–690 (2009).
24. Waraho, D. & DeLisa, M. P. Versatile selection technology for intracellular protein-protein interactions mediated by a unique bacterial
hitchhiker transport mechanism. Proc. Natl. Acad. Sci. USA 106,
3692–3697 (2009).
25. Brizzard, B. L., Chubet, R. G. & Vizard, D. L. Immunoaffinity purification of FLAG epitope-tagged bacterial alkaline phosphatase
using a novel monoclonal antibody and peptide elution. Biotechniques 16, 730–735 (1994).
26. Wegner, G. J., Lee, H. J. & Corn, R. M. Characterization and optimization of peptide arrays for the study of epitope-antibody interactions using surface plasmon resonance imaging. Anal. Chem. 74,
5161–5168 (2002).
27. Ranawakage, D. C., Takada, T. & Kamachi, Y. HiBiT-qIP, HiBiT-based
quantitative immunoprecipitation, facilitates the determination of
antibody affinity under immunoprecipitation conditions. Sci. Rep. 9,
6895 (2019).
28. Lim, B. N. et al. Directed evolution of nucleotide-based libraries
using lambda exonuclease. Biotechniques 53, 357–364 (2012).
29. Lin, J., Wang, W.-J., Wang, Y., Liu, Y. & Xu, L. Building endogenous
gene connections through RNA self-assembly controlled CRISPR/
Cas9 function. J. Am. Chem. Soc. 143, 19834–19843 (2021).
30. Zhao, E. M. et al. RNA-responsive elements for eukaryotic translational control. Nat. Biotechnol. 40, 539–545 (2022).
31. Kaseniit, K. E. et al. Modular, programmable RNA sensing using
ADAR editing in living cells. Nat. Biotechnol. 41, 482–487 (2023).
32. Ye, J.-D. et al. Synthetic antibodies for specific recognition and
crystallization of structured RNA. Proc. Natl. Acad. Sci. USA 105,
82–87 (2008).
33. Koldobskaya, Y. et al. A portable RNA sequence whose recognition
by a synthetic antibody facilitates structural determination. Nat.
Struct. Mol. Biol. 18, 100–106 (2011).
34. Koirala, D., Lewicka, A., Koldobskaya, Y., Huang, H. & Piccirilli, J. A.
Synthetic antibody binding to a preorganized RNA domain of
hepatitis C virus internal ribosome entry site inhibits translation.
ACS Chem. Biol. 15, 205–216 (2020).
Nature Communications | (2023)14:7256
https://doi.org/10.1038/s41467-023-42802-5
35. Midelfort, K. S. et al. Substantial energetic improvement with
minimal structural perturbation in a high affinity mutant antibody. J.
Mol. Biol. 343, 685–701 (2004).
36. Ellefson, J. W. et al. Directed evolution of genetic parts and circuits
by compartmentalized partnered replication. Nat. Biotechnol. 32,
97–101 (2014).
37. Guo, P., Yang, J., Huang, J., Auguste, D. T. & Moses, M. A. Therapeutic genome editing of triple-negative breast tumors using a
noncationic and deformable nanolipogel. Proc. Natl. Acad. Sci. USA
116, 18295–18303 (2019).
38. Hamilton, J. R. et al. Targeted delivery of CRISPR-Cas9 and transgenes enables complex immune cell engineering. Cell Rep. 35,
109207 (2021).
39. Hirosawa, M. et al. Cell-type-specific genome editing with a
microRNA-responsive CRISPR-Cas9 switch. Nucleic Acids Res. 45,
e118 (2017).
40. Meyer, A. J., Segall-Shapiro, T. H., Glassey, E., Zhang, J. & Voigt, C.
A. Escherichia coli ‘Marionette’ strains with 12 highly optimized
small-molecule sensors. Nat. Chem. Biol. 15, 196–204 (2019).
41. Taylor, N. D. et al. Engineering an allosteric transcription factor to
respond to new ligands. Nat. Methods 13, 177–183 (2016).
42. Spöring, M., Finke, M. & Hartig, J. S. Aptamers in RNA-based
switches of gene expression. Curr. Opin. Biotechnol. 63,
34–40 (2020).
43. Townshend, B., Xiang, J. S., Manzanarez, G., Hayden, E. J. & Smolke,
C. D. A multiplexed, automated evolution pipeline enables scalable
discovery and characterization of biosensors. Nat. Commun. 12,
1437 (2021).
44. Kim, J., McFee, M., Fang, Q., Abdin, O. & Kim, P. M. Computational
and artificial intelligence-based methods for antibody development. Trends Pharmacol. Sci. 44, 175–189 (2023).
45. Langan, R. A. et al. De novo design of bioactive protein switches.
Nature 572, 205–210 (2019).
46. Rihtar, E. et al. Chemically inducible split protein regulators for
mammalian cells. Nat. Chem. Biol. 19, 64–71 (2022).
47. Bertschi, A., Wang, P., Galvan, S., Teixeira, A. P. & Fussenegger,
M. Combinatorial protein dimerization enables precise multiinput synthetic computations. Nat. Chem. Biol. 19, 767–777
(2023).
48. Meyer, A. J., Ellefson, J. W. & Ellington, A. D. Directed evolution of a
panel of orthogonal T7 RNA polymerase variants for in vivo or
in vitro synthetic circuitry. ACS Synth. Biol. 4, 1070–1076 (2015).
49. Pu, J., Dewey, J. A., Hadji, A., LaBelle, J. L. & Dickinson, B. C. RNA
polymerase tags to monitor multidimensional protein-protein
interactions reveal pharmacological engagement of Bcl-2 proteins.
J. Am. Chem. Soc. 139, 11964–11972 (2017).
50. Shutt, T. E. & Gray, M. W. Bacteriophage origins of mitochondrial
replication and transcription proteins. Trends Genet. 22,
90–95 (2006).
51. Dubuc, E. et al. Cell-free microcompartmentalised transcriptiontranslation for the prototyping of synthetic communication networks. Curr. Opin. Biotechnol. 58, 72–80 (2019).
52. Zhang, C., Liu, H., Li, X., Xu, F. & Li, Z. Modularized synthetic biology
enabled intelligent biosensors. Trends Biotechnol. 41, 1055–1065
(2023).
53. Rivera, V. M. et al. A humanized system for pharmacologic control
of gene expression. Nat. Med. 2, 1028–1032 (1996).
54. Yoshihara, K. et al. The landscape and therapeutic relevance of
cancer-associated transcript fusions. Oncogene 34, 4845–4854
(2015).
55. Doench, J. G. et al. Optimized sgRNA design to maximize activity
and minimize off-target effects of CRISPR-Cas9. Nat. Biotechnol.
34, 184–191 (2016).
56. Sudmant, P. H., Lee, H., Dominguez, D., Heiman, M. & Burge, C. B.
Widespread accumulation of ribosome-associated isolated 3’ UTRs
10
Article
57.
58.
59.
60.
61.
62.
63.
64.
in neuronal cell populations of the aging brain. Cell Rep. 25,
2447–2456.e4 (2018).
Dhamija, S. et al. A pan-cancer analysis reveals nonstop extension
mutations causing SMAD4 tumour suppressor degradation. Nat.
Cell Biol. 22, 999–1010 (2020).
Multhoff, G. Heat shock protein 70 (Hsp70): membrane location,
export and immunological relevance. Methods 43, 229–237 (2007).
Friedrich, L. et al. Bacterial production and functional characterization of the Fab fragment of the murine IgG1/lambda monoclonal
antibody cmHsp70.1, a reagent for tumour diagnostics. Protein Eng.
Des. Sel. 23, 161–168 (2010).
Kaelin, W. G. Jr. Common pitfalls in preclinical cancer target validation. Nat. Rev. Cancer 17, 425–440 (2017).
Huovinen, T. et al. Primer extension mutagenesis powered by
selective rolling circle amplification. PLoS ONE 7, e31817 (2012).
Liu, B., Long, S. & Liu, J. Improving the mutagenesis efficiency of the
Kunkel method by codon optimization and annealing temperature
adjustment. N. Biotechnol. 56, 46–53 (2020).
Mali, P. et al. RNA-guided human genome engineering via Cas9.
Science 339, 823–826 (2013).
Mirdita, M. et al. ColabFold: making protein folding accessible to all.
Nat. Methods 19, 679–682 (2022).
Acknowledgements
We thank Dr. Yoshihiko Fujita (Kyoto University), Dr. Tatsuyuki Yoshii
(Kyoto University), Dr. Shin Kaneko (Kyoto University), Dr. Atsutaka Minagawa (Kyoto University), and Dr. Yoshihiro Shimizu (RIKEN) for helpful
discussions, and Dr. Shunsuke Kawasaki (Kyoto University) and Dr. Moe
Hirosawa (Kyoto University) for supplying materials. Dr. Kanae Mitsunaga
(Kyoto University) for technical assistance, Dr. Kelvin K. Hui (Kyoto University) and Maya Lopez (Imperial College London) for proofreading the
manuscript, and Yuko Kono, Hiromi Takemoto, and Yoshiko Ogawa for
their administrative support, and Dr. Dan Liu (National Institute of
Technology, Ariake College) for encouragement throughout the
research process. S.K. was supported by Scholarships from Iwadare
Scholarship Foundation and Honjo International Scholarship Foundation. This work was supported by JST SPRING, Grant Number
JPMJSP2110. This work was also supported by the JSPS KAKENHI (Grant
Numbers JP20H05626 and 20H05701) and iPS Cell Research Fund from
Center for iPS Cell Research and Application, Kyoto University.
Nature Communications | (2023)14:7256
https://doi.org/10.1038/s41467-023-42802-5
Author contributions
S.K. performed all the experiments. S.K., H.O., and H.S. designed
experimental strategies and wrote the paper.
Competing interests
The authors declare no competing interests.
Additional information
Supplementary information The online version contains
supplementary material available at
https://doi.org/10.1038/s41467-023-42802-5.
Correspondence and requests for materials should be addressed to
Hirohide Saito.
Peer review information Nature Communications thanks Kirill Alexandrov, and the other, anonymous, reviewer(s) for their contribution to the
peer review of this work. A peer review file is available.
Reprints and permissions information is available at
http://www.nature.com/reprints
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Open Access This article is licensed under a Creative Commons
Attribution 4.0 International License, which permits use, sharing,
adaptation, distribution and reproduction in any medium or format, as
long as you give appropriate credit to the original author(s) and the
source, provide a link to the Creative Commons licence, and indicate if
changes were made. The images or other third party material in this
article are included in the article’s Creative Commons licence, unless
indicated otherwise in a credit line to the material. If material is not
included in the article’s Creative Commons licence and your intended
use is not permitted by statutory regulation or exceeds the permitted
use, you will need to obtain permission directly from the copyright
holder. To view a copy of this licence, visit http://creativecommons.org/
licenses/by/4.0/.
© The Author(s) 2023
11
...