Reverse transcriptases are essential for synthesizing complementary DNA (cDNA) strands from RNA templates. A deeper understanding of the properties of reverse transcriptases and their implications in cDNA synthesis is vital to the success of molecular biology experiments.
Reverse transcriptases are enzymes composed of distinct domains that exhibit different biochemical activities. RNA-dependent DNA polymerase activity and RNase H activity are the predominant functions of reverse transcriptases, although depending on the source organisms there are variations in functions, including, for example, DNA-dependent DNA polymerase activity. As shown in Figure 1, the reverse transcription process typically involves these steps:
Figure 1. Reverse transcription process.
As mentioned above, one of the intrinsic properties of reverse transcriptases is the RNase H activity. RNase H cleaves the RNA template of the RNA:cDNA hybrid concurrently with polymerization (Figure 2) [1]. The RNase H activity is undesirable for synthesis of long cDNAs because the RNA template may be degraded before completion of full-length reverse transcription. The RNase H activity may also lower reverse transcription efficiency, presumably due to its competition with the polymerase activity of the enzyme.
Figure 2. RNase H activity of reverse transcriptases on cDNA synthesis.
To improve cDNA synthesis, the RNase H activity of reverse transcriptases has been reduced or diminished by introducing mutations into the RNase H domain of the enzyme. Such mutations often result in higher yield and synthesis of longer cDNAs (Figure 3).
Figure 3. Effects of RNase H activity on cDNA synthesis. mRNAs of 9.5 kb, 7.5 kb, and 5.2 kb were reverse transcribed in duplicate reactions, using reverse transcriptases with or without RNase H activity. The results demonstrate that the RNase H– reverse transcriptase was more efficient for producing full-length cDNA. M = Marker.
The ability of a reverse transcriptase to withstand high temperatures is an important aspect of cDNA synthesis. Elevated reaction temperatures help denature RNA with strong secondary structures and/or high GC content, allowing reverse transcriptases to read through the sequence. As a result, reverse transcription at higher temperatures enables full-length cDNA synthesis and higher yields, which leads to better representation of an RNA population by the cDNAs [2,3].
Wild-type AMV reverse transcriptase displays higher thermostability than wild-type MMLV reverse transcriptase, with their optimal temperatures at 42–48°C and 37°C, respectively. Some engineered MMLV reverse transcriptases are modified to withstand temperatures up to 55°C with no discernible effects on reverse transcription efficiency (Figure 4). Such highly thermostable reverse transcriptases are especially suitable to synthesize cDNA from GC-rich RNA templates.
Figure 4. Reverse transcription at elevated temperatures. RNA templates of varying lengths were reverse-transcribed at different temperatures, using a highly thermostable, engineered MMLV reverse transcriptase. RNA templates were removed by NaOH treatment, the resulting cDNAs were analyzed by denaturating agarose gel electrophoresis, and reaction products visualized with the Invitrogen SYBR Gold Nucleic Acid Gel Stain. The results indicate thermostability of the enzyme with 100% activity even at 56.4°C.
With gene-specific primers in one-step RT-PCR, reverse transcription at higher temperatures enhances specificity of the primers’ binding to the target. This strategy enables increased yield and reduced background in subsequent PCR (Figure 5), making reverse transcriptase with increased thermostability desirable for cDNA synthesis.
Figure 5. Reverse transcription at an elevated temperature enhances PCR specificity. mRNA for four different genes was reverse-transcribed at different temperatures using oligo (dT)20 (12.3 kb mRNA) or gene-specific primers (9.3 kb, 7 kb, and 5.5 kb mRNAs). Reverse transcription at 55°C generated higher specificity of the desired targets in RT-PCR.
The processivity of a reverse transcriptase refers to the number of nucleotides incorporated in a single binding event of the enzyme. Therefore, a highly processive reverse transcriptase can synthesize longer cDNA strands in a shorter reaction time (Figure 6). Some engineered MMLV reverse transcriptases can add as many as 1,500 nucleotides in a single binding event, which represents a processivity that is about 65 times greater than that of wild-type MMLV reverse transcriptase [4].
Figure 6. The processivity of a reverse transcriptase can impact overall cDNA length.
Reverse transcriptase processivity is also associated with its affinity for the template. As such, reverse transcriptases with high processivity are resistant to common inhibitors that may have carried over from the RNA sources. Examples of reverse transcriptase inhibitors include heparin and bile salts from blood and stool, humic acid and polyphenols from soil and plants, and formalin and paraffin from formalin-fixed, paraffin-embedded (FFPE) samples. These inhibitors often remain bound to RNA and/or reduce polymerization activity [5]. Highly processive reverse transcriptases are better able to overcome such inhibition (Figure 7).
Learn about reverse transcriptase inhibitors, their inhibitory mechanisms, and tips on overcoming inhibitors in reverse transcription.
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Highly processive reverse transcriptases also perform better with RNA samples of low quality and quantity. This attribute makes highly processive reverse transcriptases ideal for RNA isolated from plant and animal tissues as well as clinical research samples, which tend to be degraded due to processing and RNase-rich environments. Likewise, these enzymes are a good choice for experiments when limited amounts of RNA are available.
Figure 7. Performance of highly processive reverse transcriptases in synthesis of cDNA from RNA samples with inhibitors or degraded RNA. (A) Reverse transcription was carried out with RNA ladders spiked with common enzyme inhibitors of biological sources (e.g., formalin from FFPE samples, hematin and heparin from blood, bile salts from blood and feces). Synthesized cDNA was analyzed by alkaline gel electrophoresis using Invitrogen SYBR Gold Nucleic Acid Gel Stain. The reverse transcriptase with high processivity (H) displays higher efficiency in cDNA synthesis than enzymes with low processivity (L1–L4). (B) Total RNA purified from different plant sources was assessed for integrity by gel electrophoresis. High-quality RNA (RIN >8) shows distinct rRNA bands, whereas degraded RNA (RIN 1–3) mainly consisted of smears and/or smaller RNA fragments. The degraded RNA was reverse-transcribed with random hexamers, using reverse transcriptases of high processivity (H) or low processivity (L3, L4). qPCR was performed with the resulting cDNA, for specific gene targets. The RT efficiency was determined by normalizing all Ct values to the Ct values of the reverse transcriptase with high processivity. H = high processivity, L = low processivity, RIN = RNA Integrity Number.
The fidelity of reverse transcriptase represents sequence accuracy maintained by the enzyme during synthesis of DNA from RNA. Fidelity is inversely correlated to the error rate of reverse transcription. MMLV-based reverse transcriptases are reported to have an error rate in the range of one in 15,000 to 27,000 nucleotides synthesized, with AMV reverse transcriptase displaying an even higher error rate [6,7].
Reverse transcriptase’s fidelity may play a significant role in applications such as RNA sequencing where sequence accuracy is critical. For most other cDNA applications, the number of incorporated errors during reverse transcription is likely negligible for two reasons: the majority of genes are shorter than 10 kb and the reverse transcription process does not amplify introduced errors in the cDNA.
Reverse transcriptases may display terminal deoxynucleotidyl transferase (TdT) activity, which results in non-template-directed addition of nucleotides to the 3′ end of the synthesized DNA. TdT activity occurs only when the reverse transcriptase reaches the 5′ end of the RNA template, adds 1–3 extra nucleotides to the cDNA end, and exhibits specificity towards double-stranded nucleic acid substrates (e.g., DNA:RNA in the first-strand cDNA synthesis and DNA:DNA in the second-strand cDNA synthesis). The non–template-directed nucleotide addition commonly occurs with the preference in the order of A>G≥C>T [8].
Different reverse transcriptases possess varying degrees of intrinsic TdT activity. With wild-type MMLV and AMV reverse transcriptases, 25–90% of synthesized DNA strands may contain extra nucleotides at the 3′ end. Engineered MMLV reverse transcriptases, on the other hand, tend to display reduced intrinsic TdT activity. In addition to reverse transcriptase choice, the rate of nucleotide addition depends on reaction conditions such as the RNA:enzyme ratio, enzyme amount, incubation time, and reaction temperature.
In general, TdT activity is undesirable because the added nucleotides do not correspond to the template. However, for certain applications such as full-length cDNA cloning, rapid amplification of cDNA ends (RACE), and RNA sequencing (RNA-Seq), reverse transcriptases may be induced to intentionally add a string of Cs to the 3′ end of the cDNA. This type of TdT activity can be triggered during, or in a later phase of, cDNA synthesis by high concentrations of magnesium and/or manganese ions [9,10]. Combined with a specially designed DNA oligo with a string of 3′ Gs (called a template-switching oligo), TdT activity can specifically modify the 3′ cDNA end and the 5′ RNA end (Figure 8). Examples of these sequence modifications include the introduction of a restriction site for subsequent cDNA cloning steps and/or addition of adapters for downstream RNA sequencing steps [11–13].
