Understanding molecular evolution
The idea of using rational design criteria to improve enzyme performance is limited by our knowledge of polymerase fine structure and function. This limitation can be overcome by mimicking nature and using directed evolution to improve enzyme properties.
Our proprietary technique, compartmentalized ribosome display (CRD), allows for fast and efficient in vitro evolution of RTs [1, 2]. The technique has enabled the introduction and selection of multiple favorable mutations in wild type MMuLV RT, resulting in new, highly thermostable and processive RTs that supersede their wild type counterparts.
Molecular evolution by the CRD technique comprises several steps (Figure 9). First, beginning with the wild type MMuLV RT gene (1), an mRNA library is created based on random mutagenesis (2). Next, the mRNA library is translated in vitro to proteins that are associated with their mRNA progenitors (3). Then, the protein–mRNA complexes are placed into RT reaction mixtures and emulsified, yielding compartments containing one protein–mRNA complex each. Finally, the temperature is increased to create selective pressure under which only the improved mutants survive and produce full-length cDNA (4). By combining the best-performing mutations, highly processive MMuLV RT mutants capable of full-length cDNA synthesis at high temperatures were constructed.
- Baranauskas A et al. (2012) Generation and characterization of new highly thermostable and processive M-MuLV reverse transcriptase variants. PEDS. doi:10.1093/protein/gzs034
- Skirgaila R et al. (2013) Compartmentalization of destabilized enzyme–mRNA–ribosome complexes generated by ribosome display: a novel tool for the directed evolution of enzymes. PEDS. doi:10.1093/protein/gzt017
Figure 9. Key steps in the molecular evolution of MMuLV RT.
Understanding RNase H activity
Wild type MMuLV RT possesses an RNA-dependent and DNA-dependent polymerase activity, and also an RNase H activity. The RNase H activity degrades RNA from RNA–DNA duplexes to allow efficient synthesis of dsDNA. However, with long mRNA templates, the RNA may be degraded prematurely, resulting in truncated cDNA. Hence, it is generally beneficial to minimize RNase H activity when aiming to produce long transcripts for cDNA cloning (Figure 10).
In contrast, RTs with intrinsic RNase H activity are often favored in qPCR applications, because they enhance the melting of RNA–DNA duplexes during the first cycles of PCR.
Figure 10. Comparison of RTs and cDNA synthesis with or without functional RNase H activity.