A phased-addition strategy enhances the yield of RNAs obtained by in vitro transcription with modified transcriptional initiators
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Date
2015-05
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The Ohio State University
Abstract
RNA is active in multiple biological processes, not only as an information carrier but in catalyzing reactions and regulating gene expression. To understand the basis for this versatility, it is critical to establish structure-function relationships in RNAs. Covalently-attached biochemical or biophysical probes (e.g., fluorophores) at site-specific positions are often used to uncover these correlates in target RNAs. Generation of such modified RNAs often requires the conjugation of molecular probes to suitable chemical functionalities that have been introduced at either terminal or internal positions in the target RNA. A simple approach to create a 5'-labeled RNA is through an in vitro transcription (IVT) using T7 RNA polymerase. Since IVTs of DNA templates containing a class III ϕ6.5 promoter by T7 RNA polymerase is preferentially initiated by GTP, guanosine analogs (nucleoside or nucleotide monophosphate) have been used as transcriptional initiators to introduce different reactive groups at the 5'-end of transcribed RNAs. Although adding guanosine analogs in a large molar excess over GTP at the start of transcription generates transcripts that are predominantly 5'-modified, the overall yield of RNA is seriously compromised due to the requirement for GTP during Based on the premise that the poor yield results from the rapid depletion of limited GTP, which was intentionally used at low concentrations to favor incorporation of the modified analog, we have investigated and validated a phased-addition strategy that results in high yields of full-length, 5'-modified RNAs from small-scale transcriptions. We examined this method using three guanosine analogs: 5'-deoxyguanosine-5'-monophosphorothioate (GSMP), 5'-deoxy-5'-hydrazinylguanosine (NH2NH-G), and 5'-azido-5'deoxyguanosine (N3-G).
The phased-addition strategy is made up of two components that differ from a typical modified in vitro transcription. The first change is that a supplementation rather than a fixed-ratio approach is used. The former consists of supplementing GTP to the IVT during the first three or four hours of transcription as opposed to the fixed-ratio approach in which no additional GTP is added once transcription is initiated. The supplementation approach allows for a second change, which is to increase the initial guanosine analog:GTP ratio without concerns about the yield. Previously reported modified in vitro transcriptions using GSMP, NH2NH-G, and N3-G were typically preformed at the following ratios: 4:1 GSMP:GTP, ~31:1 NH2NH-G:GTP, and 4:1 N3-G:GTP. The supplementation of GTP permits the use of modified analog:GTP ratios as high as 50:1. These two adjustments to a typical in vitro transcription increase the total RNA yield to levels comparable to those from a typical in vitro transcription, while permitting ~90% incorporation of the modified guanosine analog at the 5'-end.
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Keywords
RNA, In Vitro Transcription, Labeling