Directed Evolution of a Reverse Transcriptase

Biotechnology is a field that takes advantage of natural molecules with unique properties and turns them into tools for laboratory use.

A few examples of this include:

• Restriction enzymes and CRISPR-based technologies, which were borrowed from bacteria's immune defenses and adapted for precision cutting of DNA [1,2].
• Green fluorescent protein, which was originally found in jellyfish and modified for use as a visual tag in cell biology. It has since been re-engineered to create multiple versions that fluoresce different colors [3].
• Directed evolution of enzymes, a method for enhancing desirable enzymatic properties by iterative mutation and selection, which won the Nobel prize in 2018 [3].

Reverse transcription (RT) enzymes are used to convert RNA into more stable and easier-to-study complementary DNA (cDNA). One commonly used RT comes from the Moloney murine leukemia virus (M-MuLV). Scientists at Thermo Fisher Scientific needed a reverse transcriptase that was able to copy longer templates (high processivity) and to function well at higher temperatures (thermostability). To this end, they mimicked nature and used in vitro molecular evolution to improve enzyme performance [4,5]. This method—called “compartmentalized ribosome display”—follows the principles of directed evolution of enzymes, including the generation of diversity, linking genotype to phenotype, and selective pressure for desired traits (Figure 1).

Random mutations are introduced into the wild-type M-MuLV reverse transcriptase gene. This creates a large library of M-MuLV RT gene variants (Figure 1, step 2). Then, the STOP codon is removed from the end of the coding sequence. The gene variants are transcribed into messenger RNA (mRNA) in vitro. Then, ribosomes translate those mRNAs into protein, producing an array of mutant RT enzymes. Many of these mutant RT enzymes won’t work well, but a few may show improved activity.

Without a STOP codon, translation stalls and the ribosome tethers the nascent RT protein to its own mRNA. That configuration represents the “ribosome display” part of compartmentalized ribosome display. Next, the enzyme-mRNA-ribosome complexes are “compartmentalized” using an oil/water emulsion, so that every droplet contains only one mRNA/protein complex. When the reagents and buffer conditions are optimized for reverse transcription, a droplet is set up as an isolated reaction compartment (Figure 1, step 3). The oil separating the droplets prevents cross-contamination from other RT variants, thus preserving the genotype-phenotype linkage.

To select for the RT mutants with the greatest processivity and thermostability, the reaction is performed at high temperatures. The enzyme-mRNA-ribosome complexes come apart and the molecules are free to interact as catalyst and substrate in solution within each droplet. Each RT enzyme variant tries to make cDNA from its matching mRNA template. Under selective pressure, only improved mutant enzymes will succeed in producing full-length cDNA for their own gene. Only gene variants that encode RT enzymes with desired traits are amplified (Figure 1, Step 4).

 White paper: Maxima H Minus Reverse Transcriptase
 White paper: Reverse transcriptase's attributes

In summary, learning from nature and improving upon it is part of the tradition of biotechnology. Scientists at Thermo Fisher Scientific expanded on this tradition and used directed molecular evolution to identify multiple mutations which confer dramatically improved thermostability, processivity, and robust activity rates compared to wild-type M-MuLV RT. These enhanced properties support higher overall cDNA yields with a variety of templates and improved synthesis from templates with complex secondary structures. Combining high performing mutations into one gene has resulted in more powerful reverse transcriptases for RNA studies.

1. Seed KD. (2015) Battling Phages: How Bacteria Defend against Viral Attack. PLoS Pathogens 11(6): e1004847.
2. Loureiro A, da Silva GJ. (2019) CRISPR-Cas: Converting A Bacterial Defence Mechanism into A State-of-the-Art Genetic Manipulation Tool. Antibiotics 8(1):18.
3. Tsien R. (1998). The green fluorescent protein. Annual Review of Biochemistry 67:509–544.
4. Baranauskas A, Paliksa S, Alzbutas G, et al. (2012) Generation and characterization of new highly thermostable and processive M-MuLV reverse transcriptase variants. Protein Eng Des Sel 25(10):657–668.
5. Skirgaila R, Pudzaitis V, Paliksa S, et al. (2013) Compartmentalization of destabilized enzyme–mRNA–ribosome complexes generated by ribosome display: a novel tool for the directed evolution of enzymes. Protein Eng Des Sel 26(7):453–461.