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A Method for the Exponential Synthesis of RNA: Introducing the Polymerase Chain Transcription (PCT) Reaction Tingjian Chen and Floyd E. Romesberg* Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States oligonucleotides with mixed deoxy- and ribonucleotide compositions. To facilitate the exponential production of RNA, we examined reactions with a nicked DNA duplex (Figure 1). In this case, each oligonucleotide of the nicked
RNA has an ever-expanding role in biology, far surpassing acting as an intermediary in the process of information retrieval from DNA to proteins. The explosion in the biology of RNA is paralleled by an expansion in its use in academic and industrial settings, with applications ranging from affinity reagents, aptamers, and ribozymes to reagents for gene silencing and CRISPR/Cas9 editing. To develop these applications, there is also much interest in the use of modified RNA, including RNA modified at the 2′ position of the sugar, which among other things, protects it from nuclease degradation. Thus, there is a growing need to produce natural and modified RNA for development and use in these applications. Although both RNA and modified RNA may be produced via chemical synthesis, the cost of synthesis is 1−2 orders of magnitude higher than that of DNA. Thus, RNA is usually produced from the relatively inexpensive ribotriphosphates via in vitro transcription using the RNA polymerase from T7 bacteriophage (T7 RNAP). This linear amplification can produce up to 100−1000 copies of RNA per template; however, the higher levels require significant optimization and milliliter-scale reactions, and the transcription of short RNAs (∼12−24 nucleotides), which are desired for many applications, is even less efficient.1 Moreover, many sequences are inherently impossible to transcribe because of secondary structure, and the requirement for a promoter and the de novo initiation of synthesis, typically with GTP, adds sequence restrictions. Finally, the transcription of modified RNA is even more problematic because it is further limited by the substrate specificity of T7 RNAP, especially with 2′-modified nucleotides. The limitations of the conventional transcription-mediated synthesis of RNA contrast sharply with the polymerase chain reaction-mediated production of DNA, in which primers with few sequence constraints direct an exponential amplification via thermocycled reactions with a thermostable polymerase, with the elevated temperatures eliminating secondary structure. To begin to address these limitations, in 2016, we reported the use of an activity-based phage-display selection system to create a family of thermophilic DNA polymerases that can efficiently recognize nucleotides with various 2′-substituents.2 One polymerase that we produced, SFM4-3, is able to amplify oligonucleotides with 2′-F-modified nucleotides via polymerase chain reaction, and we used it to select modified aptamers that bind human neutrophil elastase.3 We were keenly interested in determining whether SFM4-3 would recognize ribonucleotides and whether the enzyme’s thermostability might bestow it with the ability to more efficiently produce RNA.4 Thermocycling a DNA template with NTPs results in the production of significant quantities of RNA, but production is not exponential, although thermocycling with combinations of dNTPs and NTPs did exponentially produce © XXXX American Chemical Society
Figure 1. Principle of polymerase chain transcription (PCT). Purple indicates DNA; orange and green indicate RNAs or modified RNAs.
strand acts as a primer to initiate RNA synthesis using the remainder of the intact strand as a template, producing a DNA/ RNA chimera. After thermal denaturation and reannealing, the RNA of a newly produced chimera is bound by the other DNA primer, which then primes transcription of its DNA. Thus, the products of transcription are themselves transcribed, resulting in the exponential production of the chimeric DNA/RNA strands, a process that we have termed polymerase chain transcription, or PCT. The DNA portion of the chimera may be retained or is easily removed by DNase I treatment (possibly leaving a 5′-terminal dN). We demonstrated that without any sequence-specific optimization, PCT was capable of producing 103−105 copies of RNA from a single strand of DNA, far in excess of what is possible with conventional transcription. Moreover, we found that PCT is especially efficient with shorter RNAs as well as with 2′-F-modified triphosphates. In addition to a massive reduction in cost and requiring only pico- to nanomolar concentrations of DNA template and the relatively inexpensive natural or modified NTPs, PCT has several additional advantages. Perhaps most notably, the thermostability of SFM4-3 allows for high-temperature transcription (temperatures of ≤72 °C were explored), making it possible to efficiently produce even highly structured RNAs. Moreover, a PCT reaction has no sequence restrictions and also facilitates 5′-labeling, only requiring the addition of the desired labeled ribonucleotide to the 3′-end of the DNA primer (Figure 2). Currently, we have demonstrated efficient “asymmetric” PCT, which produces two different RNAs. “Symmetric” PCT, using a palindromic template, appears to be more challenging because of template dimer formation during the annealing step. While we are currently working on optimizing symmetric PCT, the limitation of producing two RNAs is not expected to be Received: August 29, 2017
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DOI: 10.1021/acs.biochem.7b00846 Biochemistry XXXX, XXX, XXX−XXX
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Biochemistry
Figure 2. Production of (labeled) RNA or modified RNA via PCT and subsequent DNase I treatment. Purple indicates DNA; orange and green indicate RNAs or modified RNAs.
particularly problematic, as even if one RNA is discarded, the cost is still significantly cheaper than the cost of chemical synthesis and production of the desired RNA is still more efficient than with conventional transcription. This issue is of course eliminated altogether when multiple RNAs are desired. A more problematic limitation with PCT as currently implemented is that it is less efficient with RNAs exceeding ∼50 nucleotides in length, and while we have shown that 2′-F and 2′-azido modifications are tolerated, other modifications, such as 2′-methoxy and 2′-amino modifications, are not. These limitations likely reflect the activity and specificity of SFM4-3 itself and are currently being addressed by subjecting the enzyme to further engineering and evolution. Thus, while the current implementation of PCT should find many applications, we envision an optimized PCT that allows for the efficient exponential production of virtually any natural or modified RNA.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: fl
[email protected]. ORCID
Tingjian Chen: 0000-0002-4458-4269 Floyd E. Romesberg: 0000-0001-6317-1315 Funding
This work was supported by the Defense Advanced Research Projects Agency (DARPA; Cooperative Agreement N6600114-2-4052). Notes
The authors declare no competing financial interest.
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REFERENCES
(1) Milligan, J. F., Groebe, D. R., Witherell, G. W., and Uhlenbeck, O. C. (1987) Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res. 15, 8783−8798. (2) Chen, T., Hongdilokkul, N., Liu, Z., Adhikary, R., Tsuen, S. S., and Romesberg, F. E. (2016) Evolution of thermophilic DNA polymerases for the recognition and amplification of C2′-modified DNA. Nat. Chem. 8, 556−562. (3) Thirunavukarasu, D., Chen, T., Liu, Z., Hongdilokkul, N., and Romesberg, F. E. (2017) Selection of 2′-fluoro-modified aptamers with optimized properties. J. Am. Chem. Soc. 139, 2892−2895. (4) Chen, T., and Romesberg, F. E. (2017) Polymerase chain transcription: exponential synthesis of RNA and modified RNA. J. Am. Chem. Soc. 139, 9949−9954.
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DOI: 10.1021/acs.biochem.7b00846 Biochemistry XXXX, XXX, XXX−XXX
