Oligoribonucleotide Synthesis Using T7 RNA Polymerase and Synthetic DNA Templates
Authors and Affiliations
John F. Milligan, Duncan R. Groebe, Gary W. Witherell, and Olke C. Uhlenbeck
Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309-0215, USA
Received July 13, 1987; Revised and Accepted October 1, 1987
Abstract
The document outlines a method for synthesizing small RNAs of defined length and sequence utilizing T7 RNA polymerase along with synthetic DNA templates that contain the T7 promoter. It was found that partially single-stranded templates, which are base-paired solely in the -17 to +1 promoter region, demonstrate transcriptional efficiency comparable to linear plasmid DNA. The resulting runoff transcripts typically commence at a specific and predictable position, although they may also exhibit variability in nucleotide number at the 3' terminus. Additionally, the transcription process generates a significant amount of smaller oligoribonucleotides, ranging from 2 to 6 nucleotides in length, likely stemming from abortive initiation events. Variations in the +1 to +6 region of the promoter were observed to adversely affect transcription efficiency, yet they expand the diversity of RNAs that can be synthesized. Extensive optimization of reaction conditions has enabled the production of milligram quantities of virtually any RNA molecule that spans 12 to 35 nucleotides in length.
Introduction
Recent advancements in in vitro transcription methods driven by highly active phage polymerases signify substantial progress in RNA synthesis. The target RNA sequence must be cloned within one of the numerous available phage promoter vectors, with subsequent plasmid DNA linearization achieved through restriction enzyme activity. Under optimized conditions, runoff transcription enabled by these templates can yield hundreds to thousands of moles of RNA per mole of DNA.
Advantages of T7 RNA Polymerase
Synthetic DNA templates allow for the omission of unwanted 5' flanking sequences often introduced through cloning.
The sequence of the 3' end of the transcript does not depend upon a restriction site for template linearization.
This method circumvents time-consuming cloning, sequencing, and plasmid preparation altogether.
During investigations into the functionality of synthetic DNA templates in transcription reactions, it was established that only the promoter region must be double-stranded, leading to the revelation that a single DNA fragment can suffice for each distinct desired RNA oligonucleotide.
Materials and Methods
Synthesis and Purification of Deoxyoligonucleotides
Deoxyoligonucleotides were synthesized using an Applied Biosystems 380B DNA synthesizer, followed by purification via a 20% polyacrylamide gel electrophoresis. The purified DNA was conserved in 10 mM Tris-HCl, pH 7.0 at -20°C. Templates were produced by thermally annealing two DNA strands at 65°C for 3 minutes, followed by cooling on ice to stabilize the hybrid. The top strands (T-N) encompass the promoter region but do not act as templates, while bottom strands (B-N) contain both promoter and template sequences oriented from 3' to 5' for clarity on strand pairing.
Nucleoside Triphosphate Preparation
Nucleoside triphosphates (NTPs) were sourced from Pharmacia (P-L) Biochemicals, reconstituted to a concentration of 25 mM per NTP, adjusted to pH 8.1, and stored at -20°C. Radiolabeled NTPs were acquired from New England Nuclear Research Products, while additional labeled ATP and GTP were synthesized using a method by Johnson and Walseth. The [5'-32p] pCp was generated from [r-32P] ATP using T4 kinase.
T7 RNA Polymerase Isolation
Purified T7 RNA polymerase was isolated from E. coli strain BL21, which harbored the plasmid pAR1219, according to the protocol outlined by Davanloo et al. Furthermore, T7 RNA polymerase activity was quantified on a linearized plasmid containing the wild-type T7 promoter, yielding approximately 300,000 units per mg of protein.
Transcription Reaction Conditions
Standard reactions were performed at 40 mM Tris-HCl (pH 8.1 at 37°C), supplemented with 1 mM spermidine, 5 mM dithiothreitol, 50 µg/ml bovine serum albumin, 0.01% (v/v) Triton X-100, and 80 mg/ml polyethylene glycol (8000 MW). Varied concentrations of NTPs, MgCl2, DNA template, and T7 RNA polymerase were utilized in experimental conditions. Reactions were conducted at 37°C for designated time frames, post which RNA was denatured in loading buffer containing 7 M urea and dyes before analysis on a 20% denaturing polyacrylamide gel. Products were identified through autoradiography, isolated, and subsequently counted to determine yields.
Results
Comparison of Template Activity with T7 RNA Polymerase
Three different template types were analyzed for transcriptional activity.
Template 1: Constructed from a chemically synthesized Eco R1 fragment formed by annealing T-1 and B-1, featuring the T7 promoter and 24 base pairs downstream.
Template 2: Created by cloning Template 1 within the Eco R1 site of pUC13, providing additional downstream flanking DNA.
Template 3: Formed by annealing B-1 with an 18 nt top strand to yield a fragment with base pairing from positions -17 to +1 and a 5' overhang of 23 nucleotides.
All three templates were found to produce similar profiles of oligoribonucleotide products and the longest transcription products PL and PU were generated in equivalent quantities, indicative of effective T7 RNA polymerase activity for both plasmid and synthetic DNA templates. The ability of the synthesized products to bind R17 coat protein served as a preliminary validation of nucleotide sequence fidelity.
Analysis of RNase Digestion Products
Results from RNase digestion analysis confirmed the expected nucleotide composition of the PL and PU products. Quantitative yields of both products reflected the anticipated structure of runoff transcripts with some exhibiting extra nucleotide additions. The enzymatic hydrolysis and TLC analyses provided insights into the structural and sequence conformity of the synthesized RNAs.
Abortive Initiation Products' Importance
Examination of abortive initiation products demonstrated that an increase in the ratio of such products to full transcripts suggests potential polymerase inefficiencies or complexities associated with the initiation complex's stability. Alterations in template sequences affected the frequency and nature of abortive product formation, exposing significant insights into the mechanism of transcription initiation and elongation.
Conclusion
Utilizing T7 RNA polymerase in conjunction with synthetic DNA templates represents an efficient approach to RNA synthesis, providing an effective alternative to traditional cloning methods. This system harnesses the advantages of template size, sequence flexibility, and reaction scalability to yield pure RNA products, conducive to further biophysical and biochemical investigations.
Acknowledgments
This research was supported by the National Institutes of Health Grant GM 36944, with appreciation extended to Dr. John Dunn for his support and contributions related to T7 RNA polymerase adaptation.
References
Melton, D.A., et al. (1984) Nuc. Acids Res., 12, 4035-7056.
Chamberlin, M., Ryan, T. (1982) in "The Enzymes," Boyer, P.D., 15, 85-108.
Davanloo, P. et al. (1984) Proc. Nat. Acad. Sci., 81, 2035-2039.
McAllister, W.T. (unpublished results).
Kontani, H. et al. (1987) Nuc. Acids Res., 15, 2653-2661.
Sealey, P.G. & Southern, E.M. (1982) in "Gel Electrophoresis of Nucleic Acids," Rickwood, D. & Hames, B.D. (eds.), IRL Press, 39-75.
Johnson, R.A. & Walseth, T.F. (1979) in "Advances in Cyclic Nucleotide Research," 10, Brooker, G. et al. (eds.), 135-167.
England, T.E. & Uhlenbeck, O.C. (1978) Nature, 275, 560-561.
Chamberlin, M. & Ring, J. (1973) J. Biol. Chem., 248, 2235-2244.
Nishimura, S. (1979) in "Transfer RNA: Structure, Properties, and Recognition," Cold Spring Harbor Laboratory, 551-561.
Randerath, K. et al. (1980) in "Methods in Enzymology", 65, Academic Press Inc., 638-680.
Uhlenbeck, O.C. et al. (1983) J. Biomol. Struct. and Dyn., 1, 539-552.
Martin, C.T. & Coleman, J.E. (1987) Biochem., 26, 2690-2696.
Dunn, J.J. & Studier, F.W. (1983) J. Mol. Biol., 166, 477-535.
Ikeda, R.A. & Richardson, C.C. (1986) Proc. Nat. Acad. Sci., 83, 3614-3618.
Von Hippel, P.H. et al. (1984) Ann. Rev. Biochem., 53, 389-446.
Carpousis, A.J. & Gralla, J.D. (1980) Biochemistry, 19, 3245-3253.
Levin, J.R. et al. (1987) J. Mol. Biol., 196, 85-100.
McClure, W. (1985) Ann. Rev. Biochem., 54, 171-204.
Kwang, C. & Wu, C.W. (1987) Nuc. Acids Res., 15, 2279-2294.
Caruthers, M.H. et al. (1986) Chemica Scripta, 26, 25-30.