Limited reverse transcriptase activity of phi29 DNA polymerase

Abstract

Phi29 (Φ29) DNA polymerase is an enzyme commonly used in DNA amplification methods such as rolling circle amplification (RCA) and multiple strand displacement amplification (MDA), as well as in DNA sequencing methods such as single molecule real time (SMRT) sequencing. Here, we report the ability of phi29 DNA polymerase to amplify RNA-containing circular substrates during RCA. We found that circular substrates with single RNA substitutions are amplified at a similar amplification rate as non-chimeric DNA substrates, and that consecutive RNA pyrimidines were generally preferred over purines. We observed RCA suppression with higher number of ribonucleotide substitutions, which was partially restored by interspacing RNA bases with DNA. We show that supplementing manganese ions as cofactor supports replication of RNAs during RCA. Sequencing of the RCA products demonstrated accurate base incorporation at the RNA base with both Mn2+ and Mg2+ as cofactors during replication, proving reverse transcriptase activity of the phi29 DNA polymerase. In summary, the ability of phi29 DNA polymerase to accept RNA-containing substrates broadens the spectrum of applications for phi29 DNA polymerase-mediated RCA. These include amplification of chimeric circular probes, such as padlock probes and molecular inversion probes.

Figure 1.

Experimental procedure. Padlock probes, with various RNA substitutions (red boxes) at the 3′ terminus or in the probe backbone were circularised on synthetic RNA templates (blue) by ligation and used as circular substrates in rolling circle amplification (RCA). During RCA, replication was monitored in real-time by SYBR Gold incorporation into rolling circle products (RCPs) (green). For counting and size quantification, RCPs were further labelled with Cy3- decorator probes (orange, dotted) by hybridization to the reporter sequence (green, dotted). RCPs were then imaged and digitally quantified.

Figure 2.

Effect of RNA substitutions in circular templates on rolling circle amplification with phi29 DNA polymerase. (A) Total amount of RCA products (y-axis) generated for padlock probes with/without a terminal 3′ RNA and in the absence of synthetic RNA ligation template (template -). (B) Circles with 0–7 RNA substitutions in the backbone were amplified and digitally counted. The y-axis shows the number of rolling circle products (RCPs); error bars ± S.D.; n = 2. The same RCA reactions with chimeric circles were also monitored in real-time by measuring SYBR Gold incorporation on qPCR instrument (C and E). (C) RCA reaction curves of circles with 0, 1 and 2 RNA substitutions. (D) RCPs from C were imaged on microscope slides and size and intensity of individual RCPs were quantified. Black line, median; upper whisker, highest value that is within 1.5 the interquartile range of the hinge; lower whisker, lowest value within 1.5 the interquartile range of the hinge. (E) Real-time data of the same RCA reactions as in B with 0–7 RNA substitutes are displayed. Representative samples are presented from a duplicated experiment. To highlight the initial stages of RCA and to show the difference between the samples with low RCA efficiency, fluorescence intensity readout between 3000 and 6000 is presented.

Figure 3.

Phi29 DNA polymerase exhibits higher RCA rate with circles containing pyrimidine RNA substitutions. (A) Real-time RCA curves of circles containing 1, 2, 3 or 4 consecutive RNA substations of rG, rU, rA, rC RNA bases are displayed (number of consecutive substitutions is indicated above plots). Rate of RCA was monitored by measuring fluorescence build-up (y-axis) resulted from SYBR Gold incorporation into RCPs. Averaged fluorescence intensity for each RCA time point was calculated from a duplicated experiment. RCA was conducted in the presence of Mg²⁺ and Mn²⁺ (solid and dashed lines respectively). (B) Linear, early stage RCA velocity (y-axis) is presented for PLPs from (A) in the presence of Mg²⁺ (solid lines) and Mn²⁺ (dashed lines). (C) RCA for the control PLP (non-chimeric DNA circle, with Mg²⁺ (solid) and Mn²⁺ (dashed line) are displayed.

Figure 4.

DNA sequencing-based analysis of rolling circle products reveals reverse transcription activity of phi29 DNA polymerase. (A) After RCA, short DNA oligonucleotides were hybridized to an AluI restriction site in the RCA products and RCPs were digested with AluI restriction enzyme, resulting in RCA monomers. Following digestion, monomers were PCR-amplified using primers containing Ilumina adapter sequences. PCR products were extended using IIlumina indexed primers. Finally, sequencing library was prepared using indexed primers-specific P5/7 PCR primers. The region of interest containing RNA substitutions in the original padlock probe sequence is indicated with green boxes. (B) Logos showing sequencing frequencies for each position within RCA monomers generated from the control DNA circle (P1 = dG), and circles containing single rG, rU, rA and rC substitutions at the RNA position (P1). Positions P1 and P2 are indicated and position P1 was additionally highlighted with the red box. (C) Incorporation of incorrect nucleotides for every position in the sequenced monomers from (B). Error rates, calculated as Incorporation error [%] = 1 – number of reads with expected nucleotide/total number of reads, is presented for padlock probes with single- (upper plot) and double-RNA substitutions (lower plots). P1 position for the first RNA substitution is indicated with the box.