Enzyme Activity Measurement for RNA-Directed DNA Polymerase

Creative Enzymes is pound to provide enzyme activity assays for RNA-directed DNA polymerase. We are committed to positioning as the most reliable provider of enzyme analysis services in the global market. Today, Creative Enzymes enjoys the reputation of the leading company in the field of enzymatic assays and is well known to provide high customer satisfaction. Our prompt service, best customer care, and dedicated approaches have made us the most preferred vendor.

RNA-directed DNA polymerases (EC 2.7.7.49; reverse transcriptases; RTs) are mainly derived from retroviruses, such as avian myeloblastosis virus (AMV) or Moloney murine leukemia virus (MMLV), which use the enzymes to generate complementary DNA (cDNA) from their RNA templates, a process termed reverse transcription. However, non-retroviruses also use RTs, such as hepatitis B virus (HBV). AMV and MMLV RTs are multifunctional enzymes, but they are mainly used as RNA-directed DNA polymerases. Specifically, oligodeoxynucleotides – either oligo polymers or a collection of short, specific or random DNA sequences – are used as primers for extension on RNA templates. The DNA synthesized from the RNA template is referred to as complementary DNA (cDNA). Besides, RTs also have a DNA-directed DNA polymerase activity (Figure 1), although the incorporation of dNTPs is very slow, about 5 nucleotides per second. In addition, the DNA polymerase activity of reverse transcriptase lacks a 3’→5’ exonuclease activity, which can be advantageous for certain applications. Furthermore, the third activity of RTs degrades the RNA in an RNA:DNA hybrid (Figure 2). Functionally, the RNase H activity serves as a useful part of the retrovirus’ life cycle, but it can also selectively destroy parts of an RNA molecule to which DNA molecules have been hybridized.

Figure 1: Reverse transcriptase shows 5’→3’ DNA polymerase activity (DNA-directed DNA polymerase activity).
Reference: Tzertzinis G, Tabor S, Nichols N M. Current Protocols in Molecular Biology, 2008: 3.7. 1-3.7. 4.

Figure 2: Reverse transcriptase also shows 5’→3’ and 3’→5’ exoribonuclease activity (RNase H activity).
Reference: Tzertzinis G, Tabor S, Nichols N M. Current Protocols in Molecular Biology, 2008: 3.7. 1-3.7. 4.

RTs play a pivotal role in biological analysis, drug development, and antiviral therapy. For example, RTs are commonly used in research to apply the polymerase chain reaction technique to RNA in a technique called reverse transcription polymerase chain reaction (RT-PCR). In addition, HIV-1 RTs serve as the major targets of antiviral therapy. RTs also have been reported to be related to cancer therapeutics.

Thus, these above-mentioned functions illustrate the indispensability and significance of RT activity assays as tools for quantitation and characterization of different retroviruses. Creative Enzymes is completely qualified for providing the most reliable activity assays for RTs. The colorimetric RT assay is designed for use in research studies, as a method for the quantitative determination of RT activity in cell cultures and other biological samples. The assay has been demonstrated to be useful for determining RT activity from a variety of retroviruses, such as HIV-1, HIV-2, AMV and MMLV. Creative Enzymes is happy to learn the details of your need in activity assays of RTs, and will make sure of the proper operation of the tests.

Figure 3: The crystal structure of RT from the human immunodeficiency virus type 1.
PDB: 1HMV