Temin and Baltimore's discovery of reverse transcriptase (RT) altered the understanding of molecular biology (Temin and Mizutani, 1970; Baltimore, 1970). It demonstrated that genetic information does not flow unidirectionally, from DNA to RNA to proteins, but could also flow in the reverse direction from RNA back to DNA. RT enzymes were initially found in retroviruses (e.g., Moloney murine leukemia virus (MMLV)) but have since been discovered in other RNA elements (e.g., group II introns, transposable elements) (Boeke and Stoye, 1997) and are primarily responsible for converting an RNA genome into DNA for integration into a host's chromosome. Since its discovery, RT has revolutionized the understanding of eukaryotic biology enabling the conversion of mature mRNA into cDNA, without the introns present in genomic DNA. Since these foundational studies, the RT has become a ubiquitous tool in molecular biology driving enabling technologies like next-generation RNA-Sequencing.
All known RTs are derived from a shared common ancestor (Xiong and Eickbush, 1990). These enzymes are characteristically mesophilic and lack a proofreading domain (3′-5′ exonuclease), which is thought to be the cause of their high error rate in vitro (Roberts et al., 1988). As a result of this, insertion of the correct nucleotide is driven entirely by Watson-Crick hydrogen bonding and geometry (Kim et al., 2005). In addition, the low polymerization temperature has been a notorious issue inhibiting efficient reverse transcription due to RNAs adopting stable secondary structures at lower temperatures (Klarmann et al., 1993). In contrast to RTs, high fidelity DNA polymerases have emerged and innovated biotechnology-enabling unprecedented fidelity and high thermostability.
Monomeric archaeal Family-B polymerases (polB) have been widely adopted in modern molecular biology due to their hyperthermostability, processivity, and fidelity. These enzymes have clear advantages over RTs but they have little to no activity on RNA templates. A comparison between two common archaeal enzymes (KOD and PFU) (Takagi et al., 1997; Lundberg et al., 1991) and MMLV RT reveals the wildtype archaeal polB enzymes failed to polymerize over even five RNA bases (FIG. 1A). The DNA specificity of these polymerases has likely been driven by evolutionary pressures, as these are presumed to be the genome replicating polymerase and contain mechanisms actively precluding RNA as a substrate (Greagg et al., 1999).