A universal method to produce in vitro transcripts with homogeneous 3' ends

Abstract

A method is described that allows a general drawback of in vitro transcription assays to be overcome: RNA polymerases tend to add extra nucleotides to the RNA 3' end that are not encoded in the linearized DNA template. Furthermore, these polymerases show a considerable rate of premature termination close to the RNA's 3' end. These features lead to a decreased yield of full-length transcripts and often make it difficult to determine and isolate the correctly transcribed full-length RNA. The hammerhead ribozyme is frequently used in cis to cleave off these extra nucleotides. However, the upstream sequence requirements of this ribozyme restrict its general usability. In contrast, the hepatitis delta virus ribozyme has no such requirements and can therefore be applied to any RNA sequence in cis. Due to the catalytic activity of the ribozyme, the desired transcript is released as an RNA molecule with a homogeneous 3' end. The resulting 2',3'-cyclo-phosphate group of the released RNA can be easily and efficiently removed by T4 polynucleotide kinase treatment. The presented method can be applied for virtually any sequence to be transcribed and is therefore superior to other ribozyme strategies, suggesting possible applications in every field where transcripts with homogeneous 3' ends are required.

Figure 1

Scheme showing the construction of PCR products consisting of an RNA sequence of interest attached to the HDV ribozyme sequence. (A) Construction of the HDV ribozyme sequence starting with an overlapping primer pair. (B) Amplification of the RNA sequence of interest as a PCR product carrying the 5′ part of the ribozyme sequence. (C) HDV PCR product A is used together with PCR product B in order to create the full-length PCR product consisting of an upstream T7 promoter, the sequence of interest, and the adjacent HDV ribozyme sequence. This DNA molecule is subsequently used for in vitro transcription. Fragment length is proportional to corresponding sequences. Size and sequences are given under Materials and Methods.

Figure 2

Comparison of the conventional run-off transcription and the ribozyme mediated generation of homogeneous transcripts on a denaturing 10% polyacrylamide gel. While the run-off transcription leads to a set of products of different length, the ribozyme technique leads to one single band corresponding to the transcript of the expected size (RNA) and the released ribozyme (HDV). No premature termination products or transcripts carrying non-encoded extra nucleotides are detectable.

Figure 3

Influence of the nucleotide immediately upstream of the cleavage position. The calculated cleavage efficiencies as the ratio between the signal intensities of the released tRNA^Phe and the remaining precursor molecules show that all four nucleotides are tolerated by the ribozyme.

Figure 4

Removal of the terminal 2′,3′ cyclic phosphate group of the released tRNA resulting from the HDV ribozyme cleavage reaction. Treatment with T4 polynucleotide kinase (T4 PNK) leads to the removal of the phosphate group and therefore to a reduced net charge of the RNA. This can be observed by a reduced electrophoretic mobility on a denaturing 10% polyacrylamide gel in comparison with the untreated control (mock incubation without T4 PNK).