Methods for enzymatic synthesis of modified nucleic acids (MESNA)
Lead Research Organisation:
University of Manchester
Department Name: Chemistry
Abstract
Nucleic acids are polymers comprising of nucleotide monomers, with ATCG bases in DNA or AUCG bases in RNA (U & T are equivalent). In cells, DNA exists as a double helix and is transcribed to single-stranded messenger RNA (mRNA), which is then translated to create specific proteins (functional molecules within cells). The Pfizer and Moderna COVID-19 vaccines are mRNA sequences coding for the SARS-CoV-2 spike protein. Upon immunisation the mRNA enters our cells and is translated to produce the spike protein (antigen) leading to the production of antibodies (an immune response) that protect us from future infection. Both vaccines use modified mRNA with a synthetic monomer (N1-methylpseudouridine) in place of U. Although N1-methylpseudouridine and U code for the same information, the slight structural differences improve mRNA longevity in the cell and boost translation levels of the antigen. Similarly, modified mRNAs are also being developed to combat other diseases such as cancer (immunotherapies). Currently mRNA vaccines and therapeutics are produced using a DNA-dependant polymerase enzyme. Whilst this works well, the use of DNA templates prevent selective modification as the enzyme can only use four monomers (AUCG or equivalent). Therefore, inclusion of modifications at specific positions (e.g. terminal regions more prone to degradation) is unachievable using this method.
Other examples of therapeutically important modified nucleic acids include short antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs). ASOs bind to a complementary mRNA (base-pairing) to block it or induce cleavage, preventing translation of detrimental proteins associated with a disease (e.g. genetic disorders or cancer). siRNA are short modified double stranded RNAs that associate with proteins in the cell and promote breakdown of complementary target mRNAs. ASOs and siRNAs are highly modified to improve their stability to cellular enzymes that degrade nucleic acids. Given their highly modified structures, ASOs and siRNAs are currently produced by chemical solid-phase synthesis (SPS). Although SPS works well on a small-scale, it is extremely costly and very difficult to operate at large-scale, which means that manufacture of ASOs and siRNAs required for large patient populations is not feasible. The synthetic monomers used are also very expensive to produce and require extensive chemical manipulation. Large excesses of monomers are required at each step, along with other costly reagents, and large volumes of organic solvents, most of which are toxic, damaging to the environment and increasingly unsustainable.
In this project we will develop novel enzymatic methods for template-free assembly of modified nucleic acids (mRNA, ASOs, siRNA & other therapeutics). Enzymatic methods operate in water, under mild conditions, utilising benign enzymes and renewable monomers and will provide a more sustainable, scalable, and cost-effective alternative to SPS, while also allowing selective modification of longer mRNA. Initially, we will focus on engineering template-free polymerase and ligase enzymes to accept modified nucleotide monomers with blocking groups. A blocking group ensures only one monomer is enzymatically added in each step. Only after deblocking is the next monomer added, which gives complete control over the sequence and position of modifications. We will use X-ray (3D) structures of the enzymes to guide engineering (mutagenesis), making rational changes to the enzyme active site so that modified nucleotides are accepted. We will also use more random approaches to create larger libraries of mutant enzymes. High-throughput fluorescent assays will be developed, where incorporation of a monomer leads to fluorescence, allowing us to screen larger numbers of mutants and select variants with improved properties. The engineered ligase enzymes can also be used to join longer RNA strands to produce mRNA with selective modifications.
Other examples of therapeutically important modified nucleic acids include short antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs). ASOs bind to a complementary mRNA (base-pairing) to block it or induce cleavage, preventing translation of detrimental proteins associated with a disease (e.g. genetic disorders or cancer). siRNA are short modified double stranded RNAs that associate with proteins in the cell and promote breakdown of complementary target mRNAs. ASOs and siRNAs are highly modified to improve their stability to cellular enzymes that degrade nucleic acids. Given their highly modified structures, ASOs and siRNAs are currently produced by chemical solid-phase synthesis (SPS). Although SPS works well on a small-scale, it is extremely costly and very difficult to operate at large-scale, which means that manufacture of ASOs and siRNAs required for large patient populations is not feasible. The synthetic monomers used are also very expensive to produce and require extensive chemical manipulation. Large excesses of monomers are required at each step, along with other costly reagents, and large volumes of organic solvents, most of which are toxic, damaging to the environment and increasingly unsustainable.
In this project we will develop novel enzymatic methods for template-free assembly of modified nucleic acids (mRNA, ASOs, siRNA & other therapeutics). Enzymatic methods operate in water, under mild conditions, utilising benign enzymes and renewable monomers and will provide a more sustainable, scalable, and cost-effective alternative to SPS, while also allowing selective modification of longer mRNA. Initially, we will focus on engineering template-free polymerase and ligase enzymes to accept modified nucleotide monomers with blocking groups. A blocking group ensures only one monomer is enzymatically added in each step. Only after deblocking is the next monomer added, which gives complete control over the sequence and position of modifications. We will use X-ray (3D) structures of the enzymes to guide engineering (mutagenesis), making rational changes to the enzyme active site so that modified nucleotides are accepted. We will also use more random approaches to create larger libraries of mutant enzymes. High-throughput fluorescent assays will be developed, where incorporation of a monomer leads to fluorescence, allowing us to screen larger numbers of mutants and select variants with improved properties. The engineered ligase enzymes can also be used to join longer RNA strands to produce mRNA with selective modifications.
Technical Summary
Modified nucleic acids offer enormous opportunities for the prevention and treatment of disease. For example, the Pfizer and Moderna COVID-19 vaccines comprise mRNA with modified nucleotides that greatly improve their efficacy. Many short, highly modified oligos such as antisense oligonucleotides (ASO) and small interfering RNAs (siRNAs) have also been approved or are in advanced trials for treatment of a wide range of diseases. Currently, mRNAs are manufactured by in vitro transcription (IVT), whilst ASOs and siRNAs are produced by solid-phase synthesis (SPS), both with limitations. IVT uses a DNA template which means a modified nucleotide is incorporated throughout (e.g. pseudouridine in place of uridine) and selective modification is problematic. SPS affords control, but is expensive and wasteful requiring toxic reagents, organic solvents, as well as protecting groups, all of which are unsustainable and highly problematic to scale-up. To address this, we will develop template-independant enzymatic methods that can be used to construct ASOs, siRNAs and mRNA with selective modifications. We will engineer polymerases and ligases that accept modified nucleotide triphosphates (NTPs) and 3',5'-bisphosphate (pNp) monomers respectively, with reversible 3'-blocking groups that enable template-free (stepwise) assembly of target oligos. Engineered ligases will also be developed to join longer oligos together including mRNA fragments. In silico tools will be used to design more stable enzyme variants and X-ray structures, combined with molecular modelling, will guide targeted mutagenesis of active site residues to accommodate NTPs and pNp with 2'- and 3'-modifications. Also, epPCR will be used to identify hot spots for saturation mutagenesis before DNA shuffling is used to combine mutations. Novel fluorescent screening methods will be developed for rapid quantification of extension products, as well as advanced separation and MS methods for analysis of oligo products.