High-throughput iSpinach fluorescent aptamer-based real-time monitoring of in vitro transcription

Abstract

In vitro transcription (IVT) is an essential technique for RNA synthesis. Methods for the accurate and rapid screening of IVT conditions will facilitate RNA polymerase engineering, promoter optimization, and screening for new transcription inhibitor drugs. However, traditional polyacrylamide gel electrophoresis (PAGE) and high-performance liquid chromatography methods are labor intensive, time consuming and not compatible with real-time analysis. Here, we developed an inexpensive, high-throughput, and real-time detection method for the monitoring of in vitro RNA synthesis called iSpinach aptamer-based monitoring of Transcription Activity in Real-time (STAR). STAR has a detection speed at least 100 times faster than conventional PAGE method and provides comparable results in the analysis of in vitro RNA synthesis reactions. It also can be used as an easy and quantitative method to detect the catalytic activity of T7 RNA polymerase. To further demonstrate the utility of STAR, it was applied to optimize the initially transcribed region of the green fluorescent protein gene and the 3T4T variants demonstrated significantly enhanced transcription output, with at least 1.7-fold and 2.8-fold greater output than the wild-type DNA template and common transcription template, respectively. STAR may provide a valuable tool for many biotechnical applications related to the transcription process, which may pave the way for the development of better RNA-related enzymes and new drugs.

Keywords

In vitro transcription / RNA aptamer / T7 RNAP / Real-time detection

References

[1] Autour A, Westhof E, Ryckelynck M. iSpinach: a fluorogenic RNA aptamer optimized for in vitro applications. Nucleic Acids Res, 2016, 44: 2491-2500.

[2] Baiersdorfer M, Boros G, Muramatsu H, Mahiny A, Vlatkovic I, Sahin U, Kariko K. A facile method for the removal of dsRNA contaminant from in vitro-transcribed mRNA. Mol Ther Nucleic Acids, 2019, 15: 26-35.

[3] Burcar BT, Cassidy LM, Moriarty EM, Joshi PC, Coari KM, McGown LB. Potential pitfalls in MALDI-TOF MS analysis of abiotically synthesized RNA oligonucleotides. Origins Life Evol B, 2013, 43: 247-261.

[4] Chelliserrykattil J, Ellington AD. Evolution of a T7 RNA polymerase variant that transcribes 2′-O-methyl RNA. Nat Biotechnol, 2004, 22: 1155-1160.

[5] Chen X, Zhang D, Su N, Bao B, Xie X, Zuo F, Yang L, Wang H, Jiang L, Lin Q, Fang M. Visualizing RNA dynamics in live cells with bright and stable fluorescent RNAs. Nat Biotech, 2019, 37(11): 1287-1293.

[6] Conrad T, Plumbom I, Alcobendas M, Vidal R, Sauer S. Maximizing transcription of nucleic acids with efficient T7 promoters. Commun Biol, 2020, 3: 439.

[7] Dao NT, Haselsberger R, Khuc MT, Phan AT, Voityuk AA, Michel-Beyerle ME. Photophysics of DFHBI bound to RNA aptamer Baby Spinach. Sci Rep, 2021, 11(1): 1-9.

[8] Fernandez-Millan P, Autour A, Ennifar E, Westhof E, Ryckelynck M. Crystal structure and fluorescence properties of the iSpinach aptamer in complex with DFHBI. RNA, 2017, 23: 1788-1795.

[9] Filonov GS, Moon JD, Svensen N, Jaffrey SR. Broccoli: rapid selection of an RNA mimic of green fluorescent protein by fluorescence-based selection and directed evolution. J Am Chem Soc, 2014, 136: 16299-16308.

[10] Gholamalipour Y, Karunanayake Mudiyanselage A, Martin CT. 3' end additions by T7 RNA polymerase are RNA self-templated, distributive and diverse in character-RNA-Seq analyses. Nucleic Acids Res, 2018, 46: 9253-9263.

[11] Gong P, Martin CT. Mechanism of instability in abortive cycling by T7 RNA polymerase. J Biol Chem, 2006, 281: 23533-23544.

[12] Guzman-Zapata D, Dominguez-Anaya Y, Macedo-Osorio KS, Tovar-Aguilar A, Castrejon-Flores JL, Duran-Figueroa NV, Badillo-Corona JA. mRNA imaging in the chloroplast of Chlamydomonas reinhardtii using the light-up aptamer Spinach. J Biotechnol, 2017, 251: 186-188.

[13] Henderson KL, Evensen CE, Molzahn CM, Felth LC, Dyke S, Liao GY, Shkel IA, Record MT. RNA polymerase: step-by-step kinetics and mechanism of transcription initiation. Biochemistry, 2019, 58: 2339-2352.

[14] Hofer K, Langejurgen LV, Jaschke A. Universal aptamer-based real-time monitoring of enzymatic RNA synthesis. J Am Chem Soc, 2013, 135: 13692-13694.

[15] Imburgio D, Rong MQ, Ma KY, McAllister WT. Studies of promoter recognition and start site selection by T7 RNA polymerase using a comprehensive collection of promoter variants. Biochemistry, 2000, 39: 10419-10430.

[16] Jain S, Venkataraman A, Wechsler ME, Peppas NA. Messenger RNA-based vaccines: past, present, and future directions in the context of the COVID-19 pandemic. Adv Drug Deliv Rev, 2021, 179.

[17] Ju Y, Kim HY, Ahn JK, Park HG. Ultrasensitive version of nucleic acid sequence-based amplification (NASBA) utilizing a nicking and extension chain reaction system. Nanoscale, 2021, 13: 10785-10791.

[18] Kanavarioti A. HPLC methods for purity evaluation of man-made single-stranded RNAs. Sci Rep, 2019

[19] Kartje ZJ, Janis HI, Mukhopadhyay S, Gagnon KT. Revisiting T7 RNA polymerase transcription in vitro with the Broccoli RNA aptamer as a simplified real-time fluorescent reporter. J Biol Chem, 2021, 296.

[20] Liu QJ, Ke YQ, Kan YH, Tang XJ, Li XJ, He YJ, Wu L. Compatibility and fidelity of mirror-image thymidine in transcription events by T7 RNA polymerase. Mol Ther-Nucl Acids, 2020, 21: 604-613.

[21] Orlov MA, Ryasik AA, Sorokin AA. Destabilization of the DNA duplex of actively replicating promoters of T7-like bacteriophages. Mol Biol, 2018, 52(5): 686-692.

[22] Ouellet J. RNA fluorescence with light-up aptamers. Front Chem, 2016, 4: 29.

[23] Padmanabhan R, Sarcar SN, Miller DL. Promoter length affects the initiation of T7 RNA polymerase in vitro: new insights into promoter/polymerase co-evolution. J Mol Evol, 2020, 88: 179-193.

[24] Paige JS, Wu KY, Jaffrey SR. RNA mimics of green fluorescent protein. Science, 2011, 333: 642-646.

[25] Passalacqua LFM, Dingilian AI, Luptak A. Single-pass transcription by T7 RNA polymerase. RNA, 2020, 26: 2062-2071.

[26] Patwardhan RP, Lee C, Litvin O, Young DL, Pe'er D, Shendure J. High-resolution analysis of DNA regulatory elements by synthetic saturation mutagenesis. Nat Biotechnol, 2009, 27: 1173-1175.

[27] Paul S, Stang A, Lennartz K, Tenbusch M, Uberla K. Selection of a T7 promoter mutant with enhanced in vitro activity by a novel multi-copy bead display approach for in vitro evolution. Nucleic Acids Res, 2013, 41.

[28] Rodrigues RC, Ortiz C, Berenguer-Murcia A, Torres R, Fernandez-Lafuente R. Modifying enzyme activity and selectivity by immobilization. Chem Soc Rev, 2013, 42: 6290-6307.

[29] Steitz TA. The structural changes of T7 RNA polymerase from transcription initiation to elongation. Curr Opin Struct Biol, 2009, 19: 683-690.

[30] Su YC, Hammond MC. RNA-based fluorescent biosensors for live cell imaging of small molecules and RNAs. Curr Opin Biotech, 2020, 63: 157-166.

[31] Trachman RJ, Abdolahzadeh A, Andreoni A, Cojocaru R, Knutson JR, Ryckelynck M, Unrau PJ, Ferre-D'Amare AR. Crystal structures of the mango-II RNA aptamer reveal heterogeneous fluorophore binding and guide engineering of variants with improved selectivity and brightness. Biochemistry, 2018, 57: 3544-3548.

[32] Villicaña C, Cruz G, Zurita M. The basal transcription machinery as a target for cancer therapy. Cancer Cell Int, 2014, 14(1): 1-5.

[33] Yin YW, Steitz TA. The structural mechanism of translocation and helicase activity in T7 RNA polymerase. Cell, 2004, 116: 393-404.

[34] Ying ZM, Yuan YY, Tu B, Tang LJ, Yu RQ, Jiang JH. A single promoter system co-expressing RNA sensor with fluorescent proteins for quantitative mRNA imaging in living tumor cells. Chem Sci, 2019, 10: 4828-4833.

[35] Zhang YC, Huang QY, Deng ZX, Xu YC, Liu TG. Enhancing the efficiency of cell-free protein synthesis system by systematic titration of transcription and translation components. Biochem Eng J, 2018, 138: 47-53.

[36] Zheng G, Zhao L, Yuan D, Li J, Yang G, Song D, Miao H, Shu L, Mo X, Xu X, . A genetically encoded fluorescent biosensor for monitoring ATP in living cells with heterobifunctional aptamers. Biosens Bioelectron, 2022

Funding

2018YFE0200501

2020YFA0907900