In vitro synthesis of RNA from cloned genes or sequences is crucial to understand how a virus infects its target cells and replicate.

To study viral biology and uncover new approaches to drug viral targets, researchers must make use of staple molecular biology methods to engineer viral genes, build constructs, and express key biomolecules. When done successfully, researchers can use both wild-type and mutant viral components to collect essential virological and immunological information. Researchers may need these materials for many applications, such as receptor binding specificity investigations, enzymatic activity assays, vaccine development programs, antibody development efforts, and beyond.

The complexity of downstream studies and assays can overshadow this essential molecular biology, but engineering and producing viral biomolecules requires a lot of careful planning as well. To support labs at this stage of their research, this blog will discuss some key aspects of successfully producing viral RNA and proteins, focusing in on construct creation and recombinant gene expression. 

Creating your construct(s) of interest

To generate viral nucleic acids and/or proteins for research purposes users start by establishing their gene(s) of interest and determining their end goal. With an end goal in mind, you can determine which expression system best suits your experimental needs.[1] From there, the next step is to determine a strategy for creating your cloning construct. This step will ultimately enable you to insert a gene into your vector of choice, which pairs with your chosen expression system.

The next key decision is whether you want to generate your construct and perform cloning yourself. Obviously, this approach provides the most control and often lower costs, but construct creation and cloning can be lengthy and challenging, especially if you lack experience. For this reason, some labs opt to enlist cloning services from vendors to speed their work up.

If you decide to make your construct yourself, it’s key to select the right cloning strategy. Cloning strategies largely rely on PCR (or RT-PCR for RNA viruses) to amplify specific viral genes, but primer selection is often dictated by the sequences surrounding the gene of interest and the construct generation approach you choose. Thus, primer design can play a huge role in determining how you ultimately insert that gene into your vector. However, a wide range of cloning and construct creation approaches are out there, so the choice can be overwhelming.[2] Below, we briefly outline five common approaches.

1. Restriction enzyme-based cloning

When most people think about cloning, they envision restriction enzyme cloning, which was the first cloning approach and the historical standard.[3,4] This requires researchers to review their gene sequence and identify restriction enzyme (RE) cut sites flanking either side of their target. However, researchers must ensure that cut sites don’t exist within the gene itself. Once cut from PCR amplified products, vectors are cut with the same REs to ensure hybridization and then ligated together.

2. TA cloning

TA cloning is another common approach that reduces user dependence on specific restriction enzyme digestion. TA cloning instead uses a Taq polymerase that includes a terminal transferase activity that adds an additional deoxyadenine (dA) to the 3’ end of the PCR viral gene product.[5] This PCR product is then mixed with a vector with complimentary 3´ deoxythymidine (dT) overhangs. TA cloning offers greater simplicity and significant process speed improvement. Additionally, it works in situations that lack a clear RE strategy. One limitation of TA cloning, however, is a 50% chance of cloning your viral gene in the reverse direction.

3. Topoisomerase cloning

Topoisomerase cloning (or TOPO cloning) also obviates the need for RE and cut site identification. Instead, topoisomerase I acts as both the restriction enzyme and ligase by cutting target sequences and restabling phosphodiester bonds.[6] Researchers can use TOPO cloning with PCR products that have 3’ dA overhangs (like TA cloning) or blunt ends, but each also run the risk of inserting the sequence in the reverse direction. To get around this, many have turned to directional TOPO cloning, which specifically adds a CACC sequence to one end and enables control over gene insertion into the vector. TOPO cloning offers returns with respect to speed, especially since the ligation step is complete in as little as 5 minutes. Additionally, vector self-ligation is less likely using TOPO, thereby increasing the likelihood of success.[7]

4. Gateway cloning

Some cloning approaches additionally make use of recombination to form target constructs. Perhaps the most famous of these methods is Gateway cloning, which originated at Invitrogen.[8] Gateway cloning relies on the generation of two vectors, the entry vector, which carries the gene of interest, and destination vector, which carries the necessary information for expression and selection. Gateway cloning is driven by targeted recombination using λ-phage integrase. Flanking recombination sites are added to PCR products that enable recombination into the entry vector, which itself contains separate sites for recombination into expression vector. The main advantage of Gateway cloning is that users can quickly generate a variety of final constructs for different experiments and expression systems using the same entry clone by varying expression clones.

5. Gibson assembly

Gibson Assembly is another flexible approach that allows users to stitch multiple PCR products together using carefully constructed primer overlap schemes.[9] One PCR product primer complements a sequence on the vector, whereas the other primer complements with another PCR product primer. Using 5’-exonuclease to remove some nucleotides, complementary sequences are exposed for annealing. Then, using a polymerase to fill in the gaps and ligase to form phosphodiester bonds, the final vector is completed. Like the other approaches that do not rely heavily on REs, Gibson assembly is quick, easily connects DNA fragments together, and works well for cloning larger sequences.

Making alterations: mutagenesis and nucleic acid synthesis

Once you have your viral gene(s) of interest cloned into a specific vector, you may also want to consider tactics that introduce mutations to study biologic activities. For example, one team led by researchers at China’s National Institutes for Food and Drug Control (NIFDC) investigated over 100 different SARS-CoV-2 Spike (S) protein variants, including both naturally occurring and newly introduced mutations at putative glycosylation sites.[10] Researchers used site-directed mutagenesis PCR to generate mutant S proteins and incorporated them into pseudoviruses using a Vesicular Stomatitis Virus (VSV) backdrop to study their impact on infectivity and immunogenicity.[11]

While site-directed mutagenesis PCR is a highly popular technique, CRISPR approaches have also been successfully adopted to introduce mutations into viral genes to assist virology research.[12]

It’s also important to note that the advances in synthetic approaches have made nucleic acid synthesis increasingly viable for a wide-ranging number of applications. Instead of relying on PCR and techniques above to clone your gene of interest, you can also opt to simply generate the exact target sequence using nucleic acid synthesis technologies (e.g., GeneArt Gene Synthesis). Nucleic acid synthesis can speed up operations by eliminating time-consuming steps, while also allowing you to easily manipulate your sequences of interest and introduce specific mutations. 

Special delivery: inserting viral constructs into cellular systems

Beyond your cloning approach, you must also determine how you intend to get your genetic information into your selected cell system. The key here is match transfection approaches to your cell type.[13] Generally, speaking transfection can be broken down into non-viral and viral strategies.

Non-viral transfection methods (also known transduction & transformation) use either chemical (e.g., lipofectamine) or physical (e.g., electroporation) cell treatments. While non-viral transfection approaches can be highly versatile, they are often less efficient than viral ones.[14] 

For viral transfection, a user must select viruses that work well with their specific cell system. Researchers have historically used bacteriophages to transfer genes of interest into bacterial cells. For mammalian cells, the most common viral vectors include adenoviruses, lentivirus, retroviruses, and adeno-associated viruses, whereas baculoviruses are ideal for insect cell systems.[15] Beyond host cell susceptibility, choosing between these viral systems is largely based on the size of the genetic information, expression transience, and virus specific limitations.

Cell-free alternatives to viral RNA and protein production

In addition to cellular expression systems, it’s important to note that recent advances have made cell-free alternatives viable for producing viral RNA and proteins for a variety of purposes. Instead of relying on living cells to coordinate biosynthesis, in vitro transcription and in vitro translation strategies that instead use either cell extracts or solutions containing essential raw materials and enzymes to generate biomaterials needed for research and clinical applications. Researchers can look to COVID-19 mRNA vaccines to appreciate the power of in vitro expression approaches.[16] While these approaches can cut time-consuming transfection and cell culture steps to help produce key viral RNA and proteins faster, not all gene products can be produced this way. 

Find help expressing yourself… if you need it!

"When it comes to making the biomolecules you need for your research, there’s clearly a lot to consider. While this blog was meant to help you home in on a strategy, it’s only scratched the surface of the methods out there. Remember, if you’re not sure what methods to try or want to explore the space more fully, don’t be afraid to talk to colleagues and vendors for help.

To this end, if you want to start expressing viral RNA or protein for your research, Thermo Fisher Scientific has reagents, kits, and services to support your work. For more information and details on relevant research tools from Thermo Fisher Scientific, check out our Virology Resource Page.

References

  1. Overview of Protein Expression. Thermo Fisher Scientific. Accessed November 8, 2021.
  2. Common Cloning Applications and Strategies. Thermo Fisher Scientific. Accessed November 8, 2021.
  3. Roberts RJ. (2005) How restriction enzymes became the workhorses of molecular biology. PNAS, 102(17): 5905-5908.
  4. Restriction Enzyme Basics. Thermo Fisher Scientific. Accessed November 8, 2021.
  5. Zhou MY et al. (2000) Universal TA Cloning. Curr Issues Mol Biol, 2(1):1-7.
  6. The Technology Behind TOPO Cloning Thermo Fisher Scientific. Accessed November 8, 2021.
  7. Tirabassi R. (2014) The Magic Continues: TOPO Cloning. BiteSizeBio. Published June 20, 2014. Accessed November 8, 2021.
  8. Katzen F. (2007) Gateway recombinational cloning: a biological operating system. Expert Opinion on Drug Discovery 2(4), 571-589.
  9. Gibson DG et al. (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods, 6(5), 343-5.
  10. Li Q et al. (2020) The Impact of Mutations in SARS-CoV-2 Spike on Viral Infectivity and Antigenicity. Cell, 182,1284-1294.
  11. Nie J et al. (2020) Establishment and validation of a pseudovirus neutralization assay for SARS-CoV-2. Emerg Microbes Infect, 2020, 9(1), 680-686.
  12. Teng M et al. (2021) Latest Advances of Virology Research Using CRISPR/Cas9-Based Gene-Editing Technology and Its Application to Vaccine Development. Viruses, 13(5), 779.
  13. Cell-Specific Transfection Protocols. Thermo Fisher Scientific. Accessed November 8, 2021.
  14. Reina O. (2016) Expanding Possibilities: Why You Need to Look into Viral Transduction. BiteSizeBio. Published July 9, 2016. Accessed November 8, 2021.
  15. Viral Vectors. Thermo Fisher Scientific. Accessed November 8, 2021.
  16. Chaudhary N et al. (2021) mRNA vaccines for infectious diseases: principles, delivery and clinical translation. Nature Reviews Drug Discovery, 20, 817-838.

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