General plasmids for producing RNA in vitro transcripts with homogeneous ends

Abstract

In vitro transcripts of bacteriophage RNA polymerases (RNAPs), such as T7 RNAP, often suffer from a considerable degree of 3'-end heterogeneity and, with certain promoter sequences, 5'-end heterogeneity. For some applications, this transcript heterogeneity poses a significant problem. A potential solution is to incorporate ribozymes into the transcripts at the 5'- and/or 3'-end of the target RNA sequence. This approach has been used quite widely but has required the generation of new transcription vectors or PCR-derived templates for each new RNA to be studied. To overcome this limitation, we have created two general plasmids for producing homogeneous RNA transcripts: one encodes a 3'- hepatitis delta virus (HDV) ribozyme and the other, used in combination with a two-step PCR, allows the production of double [5'-hammerhead (HH) and 3'-HDV] ribozyme constructs. A choice of cloning and run-off transcription linearisation restriction enzyme sites ensures that virtually any RNA sequence can be cloned and transcribed from these plasmids. For all the RNA sequences tested, good yields of transcript were obtained. These plasmids provide the tools for the simple, rapid creation of new RNA-coding plasmids to produce milligram quantities of homogeneous in vitro transcripts for all applications.

Figure 1

Plasmids for synthesising RNA transcripts with homogeneous ends. (A) The 3′-HDV transcription plasmid construct; P_T7 is the bacteriophage T7 RNAP promoter sequence and HDV is the modified HDV ribozyme RNA. Unique restriction enzyme sites for RNA gene insertion (EcoRI and NheI or NcoI) and sites for template linearisation for run-off in vitro transcription reactions (DraI, XhoI, EcoRV and XbaI) are indicated. (B) pRZ transcription vector construct with the multiple cloning sites [MCS: ApaI, XmaI (SmaI), BamHI, NcoI and NheI] and multiple linearisation sites (MLS: XhoI, EcoRV, XbaI, EcoRI) indicated; HDV and P_T7 are as in (A). (C) A general two-stage PCR strategy to produce inserts coding the target RNA and hammerhead ribozyme (HH) with matched complementary sequence (Comp) and a chosen MCS restriction site (RE). A partial HH ribozyme sequence (pHH) is introduced during the first round PCR and used as a template tag in the second round of PCR. (D) The final double ribozyme pRZ-Target RNA transcription vector construct. (E) The consensus HH ribozyme sequence (15); N = any nucleotide, N′ = nucleotide complementary to N, R = purine, Y = pyrimidine, H = any nucleotide except G. The sequence is shown with the 5′-target of the hMRP sequence (black background) and complementary nucleotides (underlined) required for H1 stem formation and cleavage. The nucleotides in the hairpin of stem H3 (outline font) can bear restriction enzyme sequences that allow useful further manipulations of the DNA (described in Results). (F) The modified genomic HDV ribozyme sequence bearing an NgoMIV site in the P1 stem (black background) and an upstream NcoI site (outline lettering). The sites of ribozyme self-cleavage are marked with an arrow in the relevant parts.

Figure 2

RNA transcription reactions from the 3′-HDV and 5′-HH/3′-HDV plasmid templates. (A) Aliquots (0.5–1.0 µl) of transcription reactions from 3′-HDV templates after 3 h incubation at 37°C separated on 8% acrylamide denaturing gels. Target RNA bands are marked with an arrowhead and labelled: VA (adenovirus VA RNA_I, 155 nt) and 58 (58 nt rRNA fragment). HDV ribozyme RNA (HDV) and precursor RNA (*) bands are also indicated. (B) Transcription reactions from the pRZ-NME1 and pRZ-hMRP constructs were analysed by 5% denaturing PAGE (lanes 1 and 2, and 3 and 4, respectively). Transcription reactions were carried out at 5 mM rNTPs/20 mM MgCl₂ (lanes 1 and 3). Following transcription, reactions were adjusted to 40 mM MgCl₂ and subjected to three rounds of thermal cycling (lanes 2 and 4). The RNA species are indicated and shown schematically under the gel: (a) HH–target–HDV, (b) target–HDV, (c) HH–hMRP and (d) target RNA.