A T7 RNA Polymerase Mutant Enhances the Yield of 5'-Thienoguanosine-Initiated RNAs

Abstract

Spectroscopic methods, which are used to establish RNA structure-function relationships, require strategies for post-synthetic, site-specific incorporation of chemical probes into target RNAs. For RNAs larger than 50 nt, the enzymatic incorporation of a nucleoside or nucleotide monophosphate guanosine analogue (G analogue) at their 5'-end is routinely achieved by T7 RNA polymerase (T7RNAP)-mediated in vitro transcription (IVT) of the appropriate DNA template containing a GTP-initiating class III Φ6.5 promoter. However, when high G analogue:GTP ratios are used to bias G analogue incorporation at the 5'-end, RNA yield is compromised. Here, we show that the use of a T7RNAP P266L mutant in IVT with 10:1 thienoguanosine (^th G):GTP increased the percent incorporation and yield of 5'-^th G-initiated precursor tRNA for a net ≈threefold gain compared to IVT with wild-type T7RNAP. We also demonstrated that a one-pot multienzyme approach, consisting of transcription by T7RNAP P266L and post-transcriptional cleanup by polyphosphatase and an exonuclease, led to essentially near-homogeneous 5'-^th G-modified transcripts. This approach should be of broad utility in preparing 5'-modified RNAs.

Keywords: RNA; T7 RNA polymerase; fluorescent probes; in vitro transcription; thienoguanosine.

Figure 1

(A) Structure of guanosine triphosphate and thienoguanosine (^thG). (B) Secondary structure of pre-tRNA^Cys, the model RNA used in this study. The sequences of the 5'-leader and 3'-trailer are provided below the secondary structure.

Figure 1

(A) Structure of guanosine triphosphate and thienoguanosine (^thG). (B) Secondary structure of pre-tRNA^Cys, the model RNA used in this study. The sequences of the 5'-leader and 3'-trailer are provided below the secondary structure.

Figure 2

(A) Schematic of the expected outcomes during an IVT that has a fixed ratio of ^thG:GTP and was peformed using either T7RNAP WT or P266L mutant. This illustration depicts qualitatively the gains with respect to abortive transcription (also, see Figure S2). (B) Comparison of a typical IVT versus the one-pot multi-enzyme approach designed to eliminate 5'-GTP-initiated RNAs.

Figure 3

(A) Purity of recombinant T7RNAP used in this study, as demonstrated by SDS-PAGE [10% (w/v) polyacrylamide] analysis. (B) One µl from a 100-µl IVT performed using either T7RNAP WT (lane 1) or P266L (lane 2) was electrophoresed on a 2% (w/v) agarose gel and stained with ethidium bromide; samples examined were from post-DNase I treatment. Lane 3 has 1 µg of the final pre-tRNA^Cys 55–23 RNA that was generated using the OPME (post-IVT RppH and Xrn-1) approach. (C) Fluorescence (λ_exc, 312 nm) of 5 µM RNA obtained after IVT with T7RNAP WT (1) or P266L (2), or using the OPME method with P266L (3). (D) RNA yields (grey) and % incorporation of ^thG (purple) with T7RNAP WT (1) or P266L (2), or using the OPME method with P266L (3). Mean and standard deviation values were calculated from three independent measurements.