Generating in vitro transcripts with homogenous 3' ends using trans-acting antigenomic delta ribozyme

Abstract

In most in vitro run-off transcription reactions with T7 RNA polymerase, transcripts with heterogeneous ends are commonly obtained. Towards the goal of finding a simple and effective procedure for correct processing of their 3' ends we propose the use of trans-acting antigenomic delta ribozyme. We demonstrate that the extension of nascent transcripts with only seven nucleotides complementary to the ribozyme's recognition site, and subsequently, the removal of those nucleotides with the ribozyme acting in trans, is an efficient procedure for generating transcripts with homogenous 3' ends. This approach was tested on two model RNA molecules: an in vitro transcript of yeast tRNA(Phe) and a delta ribozyme, which processed itself during transcription. The proposed procedure is a simple alternative to the use of ribozymes as cis-cleaving autocatalytic cassettes attached to transcript 3' ends. As there is little possibility that the required additional stretch, only seven nucleotides long, enters into stable interactions with other parts of the transcripts, it can be cleaved off with high efficacy.

Figure 1

Secondary structures of trans-acting antigenomic delta ribozyme and RNA intermediate of yeast tRNA^Phe in vitro transcript with seven additional nucleotides attached to its 3′ end, which are complementary to delta ribozyme’s recognition site. Numbering of nucleotides corresponds to the wild-type RNA sequences. In the ribozyme structure base-paired segments are denoted P1 to P4. Catalytic cleavage sites are marked by filled triangles.

Figure 2

The general schemes showing two alternative procedures to obtain RNA in vitro transcripts with homogeneous 3′ ends. The abbreviation RRS denotes the seven-nucleotide long stretches that correspond to the delta ribozyme’s recognition site. These nucleotides are cleaved off from the RNA transcription intermediates in the last step of the procedure.

Figure 3

Cleavage reaction of 5′-³²P-end-labeled tRNA^Phe/7nt intermediate with trans-acting antigenomic delta ribozyme. (A) Dependence of the cleavage reaction on different ribozyme-to-substrate ratios. The reactions were carried out in the buffer Tris–HCl, pH 7.5, in the presence of 10 mM MgCl₂, the products were separated on 12% polyacrylamide gels and visualized by autoradiography. (B) Cleavage reaction kinetics with 2- and 10-fold excess of the ribozyme (left panel, filled squares and circles, respectively). The autoradiogram shows the reaction products observed with 2-fold excess of the ribozyme at times ranging from 20 s to 60 min (right panel; C, control lane, RNA incubated in the presence of 10 mM Mg²⁺ ions for 60 min, no ribozyme added; L, formamide ladder; T1, limited digestion with RNase T1). (C) Dependence of the cleavage reaction on various ribozyme and substrate concentrations at a constant 2:1 ratio.