Biotechnology is built upon turning natural molecules with peculiar powers into laboratory tools. For example, restriction enzymes and CRISPR-based technologies were borrowed from the immune defenses of bacteria, then adapted for precision cutting of DNA.[1,2] Green fluorescent protein, originally derived from jellyfish, was modified for use as a visual tag in cell biology, and has been re-engineered by researchers to create multiple versions that fluoresce different colors.[3] It’s the same story for reverse transcriptase enzymes. Reverse transcription (synthesizing DNA from RNA) is a key step in the life cycle of retroviruses. Taking advantage of this skill, molecular biologists use reverse transcriptases (RTs) to convert RNA of interest into easier-to-study and more stable complementary DNA (cDNA).

One commonly used RT comes from the Moloney murine leukemia virus (M-MuLV). Scientists at Thermo Fisher Scientific pioneered a technique to optimize this enzyme to work better for laboratory applications.[4,5] They 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. This method—called “compartmentalized ribosome display”—follows the principles of Darwinian evolution, including the generation of diversity, linking genotype to phenotype, and selective pressure for desired traits (Figure 1).

Here’s how it works

Generation of diversity

Random mutations are introduced into the wild-type M-MuLV reverse transcriptase (RT) 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.

Connection of genotype to phenotype

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.

Selective pressure for desired traits

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).

Understanding the process of molecular evolution of a reverse transcriptase

Figure 1. Understanding the process of molecular evolution of a reverse transcriptase. Step 1 begins with the wild type version of the RT enzyme. Step 2 involves creating a library of variants using random mutagenesis. Step 3 includes compartmentalized ribosomal display to screen for functional mutants linking genotype to phenotype. Step 4 reveals those improved mutants that outperform the wild type version under selective pressure. 

Summary

Learning from nature and improving upon it is part of the tradition of biotechnology. Here we describe briefly how scientists at Thermo Fisher Scientific expanded on this tradition and used directed molecular evolution. The result was to identify multiple mutations which confer dramatically improved thermostability, processivity, and robust activity rates compared to wild-type M-MuLV RT enzymes. These features support higher overall cDNA yields with a variety of templates and improved synthesis from templates with complex secondary structures. By combining the best performing mutations into one gene, scientists have access to a more powerful reverse transcriptase for their RNA-based studies.

References