Insights into the Reverse Transcriptase Activity

Next, the reverse transcriptase activities of the full-length enzymes were investigated in primer extension experiments. The set-up employed for the N-terminally truncated variants was also applied here. In short, a 5’-[³²P]-radioactively labelled DNA primer was annealed to an RNA template and elongated via the respective enzyme. Reactions were incubated at 72 °C for 30 sec, 1 min and 5 min and subsequently analysed on a denaturing PAGE-gel (Figure 45).

Figure 44. Nuclease activity. A) Hairpin structure of template and 22-nt substrate (bold). The arrow indicates the expected cleavage position. B) Nuclease activities of Taq DNA polymerases wild-type (wt), M1, M747K, M1/M747K and RT-Taq 2 as indicated. Substrate cleavage was performed at 30 °C and determined at different time points (0, 5, 15, 30, 60 min). Reaction products were separated by denaturing PAGE. S: 22-nt substrate.

The experiments corroborated the initial findings of the respective KlenTaq variants and showed increased reverse transcriptase activity for RT-Taq 2 and Taq M1/M747K compared to the parental enzymes Taq M1 and M747K. Again template-independent nucleotide addition at blunt-ended DNA was observed in case of full-length product formation.^{[72, 107]}

3.2.4 RT-PCR

The reverse transcriptase activity is essential for applications like TaqMan based real-time RT-PCR. Thus, the potential of the full-length variants in real-time RT-PCR was investigated using an MS2 bacteriophage RNA as template (Figure 46). Under the conditions studied, the generated Taq variants RT-Taq 2 and M1/M747K showed product formation approximately eight cycles earlier than the parental enzymes M1 and M747K. Expected product formation was determined via agarose gel analysis (Figure 46). Remarkably, the wild-type enzyme also exhibited a distinct reverse transcriptase activity with product formation visible after approximately 30 cycles. This is in good accordance with reported previous studies showing some reverse transcriptase activity for the Taq wild-type enzyme.^[158]

Figure 45. Primer extension experiments with Taq wild-type (wt) and variants. A) Partial primer/template sequence. B) Primer extension reactions with 5’-[³²P]-labelled 20 nt primer annealed to an RNA template. Reactions were catalysed by Taq wt (1), M1 (2), M747K (3), M1/M747K (4) and RT-Taq 2 (5). Reactions were incubated for 30 s, 1 or 5 min, as indicated.

As preceding experiments confirmed the endonuclease and reverse transcriptase activity of RT-Taq 2, the enzyme was subsequently employed in TaqMan based real-time RT-PCR (Figure 47). A 90 nt RNA stretch of human -actin mRNA was targeted and amplified from total RNA isolated from Jurkat cells. Samples were analysed in the FAM channel using a TaqMan probe consisting of an oligonucleotide conjugated to FAM at the 5’ end and a black hole quencher (BHQ-1) at the 3’ end.

Figure 46. Real-time RT-PCR performed with Taq wild-type (wt) and variants (as indicated); F.U. = fluorescence units. The fluorescence readout was based on the binding of SYBRGreen I to double-stranded DNA. A) Amplification of a 100 bp target sequence from MS2 bacteriophage RNA. B) Amplification of a 100 bp target sequence from the respective DNA template. C) Negative control using water instead of RNA.

Product formation was analysed on a 2.5 % agarose gel as depicted below, with wt in lane 1, M1 in lane 2, M747K in lane 3, M1/M747K in lane 4, and RT-Taq 2 in lane 5.

Figure 47. Detection of a 90 nt -actin transcript from extracted human total RNA (Jurkat cells) using TaqMan based real-time RT-PCR. A polymerase dilution series was employed as indicated; F.U. = fluorescence units.

length variant RT-Taq 2 were promising regarding its nuclease and reverse transcriptase activity. The mutations present in the new variants can obviously influence the endonuclease activity, as seen for Taq M1/M747K. Nevertheless, the nuclease activity of RT-Taq 2 was comparable to the activity of the parental enzymes and is sufficient for its application in TaqMan based RT-PCR. Interestingly, an overall higher reverse transcriptase activity was observed for the full-length enzymes compared to the N-terminally truncated versions in primer extension experiments. However, variants Taq M1/M747K and RT-Taq 2 still exhibit enhanced reverse transcriptase activity compared to the parental enzymes. Furthermore, the RT-Taq 2 was successfully applied in RNA detection via TaqMan based real-time RT-PCR highlighting its potential for applications in diagnostics and molecular biology. RNA detection with other sequence contexts has to be explored. Additional experiments investigating the stability or other intrinsic properties of the enzymes are also of interest for their future application.

4. Substrate Spectrum Analysis of KlenTaq Variants 4.1 Introduction

Most DNA polymerases recognize their substrates with exceptional high specificity limiting their use in biotechnology. DNA polymerases with increased substrate spectra are therefore of great interest and a focus of directed evolution. A major goal of directed evolution is the production of enzymes with properties tailored for specific applications (see chapter I 4, 5).

Especially, the evolution of Taq DNA polymerase represented a paramount aim in research, as the enzyme is employed in various biotechnological applications. Through various techniques Taq or KlenTaq mutants were evolved towards the ability to incorporate NTPs with higher efficiency than the wild-type,[195, 196, 199] to accept sugar-modified dNTPs or NTPs[214, 215] or to replicate from lesion containing templates.[72, 159, 215]. Even the acceptance of unnatural nucleotides was achieved, paving the road for the development of artificial genetic systems;

thereby increasing the information potential of RNA and DNA.^[216]

As both parental enzymes KTq M1 and M747K possess an increased substrate spectrum, the mutation combination of both was promising to yield variants with even more advanced properties. Therefore, KTq wild-type, parental enzymes M1 and M747K as well as the N-terminally truncated (KlenTaq) variants RT-KTq 1-4 were analysed regarding their substrate specificity.

4.2 Results