Rational design of oligonucleotides for enhanced in vitro transcription of small RNA

Abstract

All kinds of RNA molecules can be produced by in vitro transcription using T7 RNA polymerase using DNA templates obtained by solid-phase chemical synthesis, primer extension, PCR, or DNA cloning. The oligonucleotide design, however, is a challenge to nonexperts as this relies on a set of rules that have been established empirically over time. Here, we describe a Python program to facilitate the rational design of oligonucleotides, calculated with kinetic parameters for enhanced in vitro transcription (ROCKET). The Python tool uses thermodynamic parameters, performs folding-energy calculations, and selects oligonucleotides suitable for the polymerase extension reaction. These oligonucleotides improve yields of template DNA. With the oligonucleotides selected by the program, the tRNA transcripts can be prepared by a one-pot reaction of the DNA polymerase extension reaction and the transcription reaction. Also, the ROCKET-selected oligonucleotides provide greater transcription yields than that from oligonucleotides selected by Primerize, a leading software for designing oligonucleotides for in vitro transcription, due to the enhancement of template DNA synthesis. Apart from over 50 tRNA genes tested, an in vitro transcribed self-cleaving ribozyme was found to have catalytic activity. In addition, the program can be applied to the synthesis of mRNA, demonstrating the wide applicability of the ROCKET software.

Keywords: ROCKET; in vitro transcription; oligonucleotide design; tRNA; thermodynamic parameters.

FIGURE 1.

Schematic illustrations of the preparation of double-stranded DNA template for in vitro RNA transcription. (A) Preparation of a DNA template from plasmid DNA. A target DNA fragment harboring the T7 promoter sequence (T7 pro) at the 5′ end of the gene is prepared from plasmid DNA by linearization using a restriction enzyme or by PCR amplification. (B) Three distinct strategies to prepare a DNA template from two oligonucleotides. Strategy 1: A double-stranded DNA template is prepared by annealing nontemplate strand (NTS) and template strand (TS) DNA oligonucleotides encoding the T7 pro sequence and the entire target gene. Strategy 2: A hemi-duplexed DNA template is prepared by annealing a NTS oligonucleotide encoding the T7 pro sequence to a TS oligonucleotide encoding the T7 pro and the entire gene. Strategy 3: Two oligonucleotides are designed to possess an overlapping region that facilitates their annealing and are extended by DNA polymerase.

FIGURE 2.

Overview of the ROCKET software. (A) Cloverleaf structures of Thermoplasma acidophilum tRNA^Leu, Thermus thermophilus tRNA^Pro, and T. kodakarensis tRNA^His. (B) Workflow for computational design of DNA oligonucleotides for in vitro transcription. (C) Oligonucleotide selection for T. acidophilum tRNA^Lue. The number of usable oligonucleotides is narrowed down in ROCKET in subsequent steps. (D) The ΔG_total (ΔG_{forward oligo} + ΔG_{forward oligo}) was calculated for each set of forward/reverse oligonucleotides. The suboptimal oligonucleotide set with the lowest (dark-gray) and the optimal set with the highest ΔG_total value (orange) are highlighted.

FIGURE 3.

Oligonucleotides selected by ROCKET enhance transcription yields. (A) Secondary structures of T. kodakarensis tRNA^His with nucleotides highlighted in blue that form the annealing region for published forward/reverse oligonucleotides (left) or for oligonucleotide pairs selected by ROCKET as optimal (right) or suboptimal (center). (B) Transcription yields from the one-pot reaction of DNA extension and RNA transcription were monitored by staining of RNA with Toluidine blue after 10% denaturing PAGE (7 M urea) (left) and quantified (right), as described in the Materials and Methods. Errors (x), SD, and P-values are shown. (C) In vitro transcription yields with comparable amounts of template DNA. Errors, SD (n = 3), and P-values are shown.

FIGURE 4.

Characterization of the DNA extension reaction. (A) Duplex-formation of published (black), suboptimal (green), and optimal (light green) oligonucleotide pairs. Numbers were obtained by quantification of electrophoretic mobility shift assays (EMSAs) shown in Supplemental Figure S4; SD (n = 3) are shown for each data point. (B) Relative DNA yields obtained in DNA polymerase extension reactions were quantified as described in the Materials and Methods. Errors, SD (n = 6), and P-values are shown. (C) PCR cycle-dependent amounts of extended DNA obtained by DNA polymerase reactions in the presence of 1 µM oligonucleotides and ∼10 nM Dream-Taq DNA polymerase (0.01 units) were quantified. The data were fitted to a single exponential curve. Errors, SD (n = 3) are shown. (D) The DNA template after 10 cycles of the DNA polymerase extension reaction was analyzed. After the DNA purification steps, DNA concentration was quantified by UV absorbance. Errors, SD (n = 3) are shown. (E) Time-dependent amounts of extended DNA obtained by DNA polymerase reactions in the presence of 1 µM oligonucleotides and ∼10 nM Dream-Taq DNA polymerase (0.01 units) were quantified. Errors, SD (n = 3) are shown. (F) DNA yields after 25 cycles of the extension reaction in the presence of ∼10 nM and ∼250 nM Dream-Taq DNA polymerase were analyzed after DNA purification steps. DNA concentration was quantified by UV absorbance. Errors, SD (n = 3) are shown.

FIGURE 5.

Characterization of the transcription reaction. (A) RNA yields obtained by in vitro transcription at specific time points were analyzed by Toluidine blue staining after 10% denaturing PAGE and (B) relative band intensities were quantified as described in the Materials and Methods and fitted to a single exponential curve (Equation 4). Errors, SD (n = 3) are shown.

FIGURE 6.

In vitro RNase P reaction on precursor tRNA. (A) A cloverleaf structure of precursor tRNA. (B–D) Cleavage of the leader sequence by RNase P. Based on optimal, ROCKET-selected oligonucleotides precursors for (B) tRNA^Gln-CTG, (C) tRNA^Gln-TTG, and (D) tRNA^iMet-CAT were in vitro synthesized and subsequently incubated with (+) or without RNase P (−). The RNAs were visualized by Toluidine blue staining after 10% denaturing PAGE (7 M urea). The asterisk marks tRNAs used as a migration control.

FIGURE 7.

In vitro transcription of all T. kodakarensis tRNA genes. DNA templates were based on tRNA genes retrieved from the genomic tRNA database (Chan and Lowe 2016) and constructed with oligonucleotides selected as optimal by the ROCKET software (Supplemental Table S1). RNAs were visualized by Toluidine blue staining after 10% denaturing PAGE of 5 µL transcription mixture. Nucleotides at positions 1 and 72 in those tRNAs that do not have a G residue as the 5′ end (marked with an asterisk), have been substituted with G1-C72.

FIGURE 8.

Comparison of oligonucleotide selection by ROCKET and Primerize. (A) Forward and reverse oligonucleotides selected by ROCKET (top) and Primerize (bottom) for T. kodakarensis tRNA^Trp with the annealing sites in color. (B) Density plot of ΔG_total values for 46 sets of oligonucleotides for T. kodakarensis tRNA genes, selected by ROCKET (blue) and Primerize (green). (C) PCR cycle-dependent amounts of extended DNA obtained by DNA polymerase reactions in the presence of 1 µM oligonucleotides and ∼10 nM Dream-Taq DNA polymerase (0.01 units) were quantified. The data were fitted to a single exponential curve. Errors, SD (n = 3) are shown. (D) Time-dependent amounts of extended DNA obtained by DNA polymerase reactions in the presence of 1 µM oligonucleotides and ∼10 nM Dream-Taq DNA polymerase (0.01 units) were quantified. Errors, SD (n = 3) are shown. (E–G) Transcription yields of (E) T. kodakarensis tRNA^His, (F) Thermoplasma acidophilum tRNA^Leu, and (G) Thermus thermophilus tRNA^Pro from one-pot reactions using Primerize-selected oligonucleotides and ROCKET-selected oligonucleotides were compared. Transcription yields were quantified by 10% denaturing PAGE (7 M urea) as described in the Materials and Methods. Errors (x) from three biological replicates, SD, and P-values are shown.

FIGURE 9.

Application of ROCKET in the preparation of catalytically active CPEB3 ribozyme. (A) Oligonucleotide sequences selected by ROCKET with the portions covering T7 promoter (T7 pro, gray), leader (dark blue), and CPEB3 ribozyme (light blue) highlighted. (B) Secondary structure of the produced transcript. The cleavage site of the CPEB3 ribozyme (between A-1 and G1) is indicated by an arrow. (C) Synthesis and self-cleavage of the CPEB3 ribozyme was monitored by 10% denaturing PAGE as described in the Materials and Methods.

FIGURE 10.

Application of ROCKET to the synthesis of EGFP protein. (A) Computational workflow for design of DNA oligonucleotides for the EGFP gene. (B) Experimental workflow for EGFP synthesis. The experiment was perfomed by two successive PCRs. The resulting template DNA was gel-purified and then used for the protein synthesis. (C) One microliter of the PCR product from each PCR step was analyzed by 2% agarose gel electrophoresis. The gel was stained by SYBR gold. (D) EGFP synthesis was monitored by fluorescence intensities where EGFP was excited by the 488 nm light, and the emission at 510 nm was measured. Errors (x) from three biological replicates, SD, and P-values are shown.

Teppei Matsuda