Reverse transcriptase is a kind of DNA polymerase that performs three different functions: RNA-directed DNA polymerase activity, RNase H activity, and DNA-directed DNA polymerase activity.

1. Synthesize the initial cDNA strand complementary to the template RNA, resulting in an RNA-DNA hybrid strand, using RNA-guided DNA polymerase activity.

2. RNase H activity hydrolyzes the RNA strand in the cDNA-RNA hybrid strand to produce single-stranded cDNA.

Note: Because RNase H may destroy the RNA template before full-length reverse transcription is accomplished, lowering or eliminating RNase H activity in reverse transcriptase improves the capacity of reverse transcriptase to create long-chain cDNA.

3. Catalyzes the synthesis of a complementary second cDNA strand from single-stranded cDNA using DNA-directed DNA polymerase activity.

Figure 1. Schematic diagram of the reverse transcription process

Reverse transcription consists of different structural enzymes with different biochemical activities. There are 6 things to note about the nature of reverse transcriptase. They are DNA polymerase activity, RNase H activity, thermostability, processivity, fidelity, and terminal nucleotidyl transferase (TdT) activity.
RNase H can simultaneously cleave RNA templates in RNA:cDNA hybrid strands during synthesis. Since the RNA template may be degraded before full-length reverse transcription is completed, RNase H enzymatic activity is a factor that needs to be avoided during the synthesis of long cDNA fragments. And because RNase H competes with the active polymerase in reverse transcriptase, RNase H enzymatic activity can reduce reverse transcription efficiency.
Factors affecting cDNA synthesis in reverse transcriptase include the ability to withstand high temperatures. Elevating the reaction temperature can denature RNAs with robust secondary structure and/or high GC content, allowing reverse transcriptase to read the sequence. Therefore, at the higher reaction temperature, reverse transcription can achieve full-length cDNA synthesis with higher yield and better reverse transcription of RNA into cDNA.
The synthetic capacity of reverse transcription refers to the number of nucleotides incorporated into a single binding site of an enzyme. Therefore, a reverse transcriptase with high synthesis capacity can synthesize longer cDNA strands in a shorter reaction time.
The fidelity of reverse transcription refers to the sequence accuracy during the reverse transcription of RNA to DNA. Fidelity and reverse transcription errors are inversely proportional.
Reverse transcriptases exhibit TdT activity only when the reverse transcriptase contacts the 5' end of the RNA template. At this point, it adds 1 to 3 additional nucleotides to the cDNA end and exhibits specificity for double-stranded nucleic acid substrates.
By developing and modulating the above intrinsic properties of reverse transcriptase, successful cDNA experiments can be performed. Not only can it be used for research its use, but also an important tool for other applications in the field of molecular biology.

 

2. What are types of reverse transcriptase?

Different reverse transcriptases have different functional activities and qualities, which impact the length, yield, and capacity to reverse complicated and challenging RNA templates, among other things. Most commercially available reverse transcriptases are derived from the avian ameloblastoma virus (AMV) or the Moloney murine leukemia virus (M-MLV). RNase H activity, reverse transcription temperature, duration, and yield vary greatly, yet each has benefits and drawbacks.

Through genetic modification based on M-MLV, Yeasen has produced Hifair™ series reverse transcriptases with reduced RNase H activity, improved thermal stability, and stronger continuous synthesis capacity to further increase reverse transcription efficiency. The features of many kinds of reverse transcriptases are listed in Table 1.

 

Figure 2. Schematic of reverse transcriptase

 

Table 1. Comparing reverse transcriptases

	AMV Reverse Transcriptase	M-MLV Reverse Transcriptase	Yeasen Hifair™ Reverse Transcriptase
Temperature	42℃	42-60℃	42-60℃
Reaction time	60 min	30-60 min	15-30 min
Yield	Middle	Low	High
Length	Short	Middle	Long，up to 19.8kb
RNase H activity	High	Middle	Low

 

3. Reverse transcriptase from the Yeasen Hifair™ series