Powerful Applications for RNA And DNA Modifying Enzymes

In this article, you will learn about RNA and DNA modifying enzymes, including polymerases, nucleases, and more. Their place in the molecular biology toolbox was solidified decades ago, but many modern-day applications are still helping to advance biological discovery, diagnostics, and therapeutics.

Basic biological questions have yielded a wide variety of cutting-edge applications for today’s molecular biologists and diagnostics or therapeutics developers. Projects that began more than 50 years ago with a simple biological question – how does bacteriophage T7 initiate a switch from early transcription genes to late transcription genes – led to the discovery of T7 RNA polymerase (RNAP). [1] Through additional work, T7 RNAP spawned the implementation of now-ubiquitous methods, such as in vitro transcription (IVT), key for many modern-day workflows and a pivotal manufacturing step for the production of Pfizer/BioNTech and Moderna’s COVID-19 mRNA vaccines. [2]

The same is true for decades-old questions about the long-term stability of genomic DNA in cells and how it endures damage from endogenous or exogenous slights. Tackling these questions revealed the molecular machinery behind base-excision, nucleotide excision, and DNA mismatch repair pathways. [3] These discoveries were major advances and led to pioneers driving the research – Tomas Lindahl, Paul Modrich, and Aziz Sancar – receiving the 2015 Nobel Prize in Chemistry. [4]
 

Similar to the examples above, basic biological questions have led to the discovery of a rich suite of additional RNA and DNA polymerases, nucleases, ligases, and more, collectively termed modifying enzymes. Since their initial discovery, a vast corpus of additional knowledge and data has been collected about these systems, driving additional innovation.

Let’s take a closer look at the role of modifying enzymes in some of biotechnology’s cutting-edge approaches.
 

Formalin-fixed paraffin-embedded (FFPE) tissue samples are a common biological sample type, collected for a broad range of oncology or immunology pre-clinical and clinical applications. After a tissue sample is collected, it’s preserved in formaldehyde and embedded in paraffin wax, thus preserving (in a semi-permanent state) the biomolecules present and the original 3-D cellular structure. The benefit of FFPE preservation is that samples can be stored for decades and analyzed later using immunohistochemistry (IHC) or other microscopy techniques. [5]

To glean genomic and transcriptomic insights, there has been increasing use of FFPE samples in NGS applications. One major challenge with this approach is that FFPE processing induces damage to nucleic acids including crosslinking (DNA-DNA or DNA/protein), fragmentation, single nucleotide polymorphisms (SNPs), and depurination, which can accumulate over time, particularly when samples are kept in suboptimal storage conditions. DNA damage at the hands of formaldehyde treatment greatly reduces the quality and quantity of DNA sequencing of both mammalian and bacterial DNA extracted from FFPE samples. [6,7]

To deal with this, researchers have turned to in vitro methodologies using a reconstituted DNA repair system that can recapitulate many of the activities seen in vivo (Figure 1). For instance, cytosine deamination to uracil is a common occurrence within FFPE samples, leading to significant, non-reproducible sequencing artifacts. Pre-treatment of extracted DNA with uracil-DNA glycosylase (UDG) – an efficient, highly-specific enzyme dedicated to removing uracil from double- and single-stranded DNA as part of the base-excision repair (BER) pathway – significantly reduces the frequency of these artifacts. [3,8,9] More complex DNA repair enzyme mixes that deal with a broader range of mammalian DNA damage, including modified bases, nicks, gaps, and more, have been developed. Their use globally reduces the amount of G:C to A:T mutations and improved single nucleotide polymorphism (SNP) and copy number variation (CNV) call rates. [10,11]

While many groups have focused exclusively on the repair of mammalian genomic DNA, characterization of FFPE-induced DNA damage and methods for repairing it would be beneficial for those doing microbial community analysis with these sample types. A recent publication, by Bueso et al. demonstrated that significant DNA crosslinking occurs in bacterial DNA (even more than in mammalian DNA) and oxidation and/or deamidation of cytosine are common forms of DNA damage, induced by FFPE treatment. [12]

The group also developed 2 repair methodologies, one for reversing DNA crosslinks and one that involved a reconstituted BER pathway. The in vitro BER reaction included treatment of extracted DNA with 3 glycosylases – formamidopyramidine DNA glycosylase (FPG), Endo VIII, and UDG – followed by repair of apurinic/apyrimidinic site or blocked ends with Endo IV or polynucleotide kinase (PNK), respectively, DNA polymerase I, and E. coli DNA ligase. Whole-genome sequencing of repaired DNA, compared to unrepaired DNA, revealed that a combination of de-crosslinking and in vitro BER increased DNA fragment length, reduced sequencing artifacts, and improved overall sequencing readability.
 

With the continuation of the SARS-CoV-2 pandemic and heightened awareness about emerging variants, sensitive and selective pathogen diagnostics are top-of-mind. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)-based methods have emerged as the gold standard for pathogen detection, particularly for SARS-CoV-2: Detection can be tailored to specific variants and detect early stages of infection.

The qRT-PCR technique requires a trained scientist and a centralized laboratory making it time-intensive, and more expensive. It can be susceptible to DNA contamination, and requires specialized instruments with multiple functions (i.e., optical capabilities and thermal cycling). As a result, there’s a need for convenient, easy-to-use point-of-care (POC) diagnostics.

One technique that has emerged as a promising alternative, which doesn't require thermal cycling or a high degree of expertise, is isothermal nucleic acid amplification. Of the various isothermal reaction types that have emerged, nucleic acid sequence-based amplification (NASBA) has broad clinical potential across a wide range of bacterial, viral, fungal, and eukaryotic pathogens. [13,14] NASBA methods detect pathogen-derived genomic RNAs in a one-pot reaction that includes reverse transcriptase (which drives first-strand and second-strand DNA synthesis of target RNA and introduces a T7 promoter sequence), RNAse H (an endonuclease that hydrolyzes the RNA portion of the RNA/DNA hybrid), and T7 RNAP (that drives transcriptional amplification of antisense target RNA). T7 RNAP-based RNA amplification is rapid (can be done in 30 minutes) and high-yield, driving the generation of up to 10¹² amplicons. [14,15] These RNAs are then quantified by annealing to a fluorescent molecular beacon probe.

Recently, a variation of the NASBA technique, called nicking and extension chain reaction system-based amplification (NESBA), was developed. As the name suggests, the technique incorporates a nicking step using a nicking endonuclease, which enables the exponential amplification of DNA, before T7 RNAP-mediated RNA amplification by in vitro transcription (IVT). The addition of this step significantly increased the sensitivity of the NASBA technique, enabling its application to clinical COVID-19 cases: In a clinical setting, NESBA accurately diagnosed 100% of 30 positive and 68 negative cases, highlighting its incredible utility as a POC diagnostic test. [15]
 

The current success of the Pfizer/BioNTech and Moderna SARS-CoV-2 vaccines has brought much attention to mRNA vaccines as a rapid and robust strategy for addressing emerging pathogens. Improving upon the current mRNA vaccine design is a major focus for vaccine developers, as the current manufacturing and distribution platform has several drawbacks, requiring ultracold storage and having a relatively short shelf life of up to 6 months. [16]

Many solutions to the stability problem have been proposed, one of which includes the formulation of mRNA vaccines as a circular, rather than a linear RNA. [17] Circular RNAs (circRNAs) have received increasing attention for their role in the normal functioning of mammalian cells as a micro RNA (miRNA) or protein sponge, scaffolding for protein complexes, and template for protein synthesis. Bioengineering of circRNAs has increased as well: Including an internal ribosome entry site (IRES) drive in vivo and in vitro production of heterologous proteins. CircRNAs are also two to five times more stable than linear RNAs, giving further support to their potential use for novel vaccines.

Biosynthetic routes of circRNAs production are an accurate and highly efficient method for production (Figure 2). [17] These methods generate linear RNA precursors through T7 RNAP-mediated IVT, followed by phosphatase treatment (to remove the 5′ triphosphate) and PNK treatment with ATP to add a 5′ monophosphate. The presence of a 5′ monophosphate and 3′ hydroxyl group enables the use of T4 DNA or RNA ligases to form circRNA. A bridging single-stranded cDNA oligonucleotide can increase the efficiency of ligation by bringing 5′ and 3′ ends together spatially. While a proof-of-concept paper, confirming the power of circRNAs vaccines, has recently been published there has yet to be a full commercial scale-up by biopharmaceutical companies. [18]
 

Figure 2. In vitro transcription workflow for generation of circRNA adopted from Obi et al. [19] (a) Double stranded DNA template for T7 RNAP-mediated IVT can be generated by PCR or by restriction digest of plasmid DNA. (b) IVT is initiated by incubating DNA template with T7 or SP6 RNAP, RNase inhibitor, and ribonucleotide triphosphates (rNTPs). (c) A 5′ monophosphate is required for circularization and can be generated by using a molar excess of GMP over GTP in the rNTP mix. Alternatively, GTP can be used to generate transcripts with a 5′ triphosphate, followed by (d) alkaline phosphatase and PNK treatment or (e) pyrophosphatase (RppH). (f) The resulting 5′ monophosphorylated RNA can then be circularized by incubation with T4 RNA ligase. A bridging DNA or RNA single-stranded oligonucleotide can be used to bring the 5′ and 3′ ends together and make the ligation reaction more efficient.
 

Given the many current applications described here and elsewhere in the scientific literature, the power and relevance of RNA and DNA modifying enzymes are clear. Explore Thermo Fisher Scientific’s large portfolio of modifying enzymes.
 

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