An enzyme cascade enables production of therapeutic oligonucleotides in a single operation.

Therapeutic oligonucleotides have emerged as a powerful drug modality with the potential to treat a wide range of diseases; however, the rising number of therapies poses a manufacturing challenge. Existing synthetic methods use stepwise extension of sequences immobilized on solid supports and are limited by their scalability and sustainability. We report a biocatalytic approach to efficiently produce oligonucleotides in a single operation where polymerases and endonucleases work in synergy to amplify complementary sequences embedded within catalytic self-priming templates. This approach uses unprotected building blocks and aqueous conditions. We demonstrate the versatility of this methodology through the synthesis of clinically relevant oligonucleotide sequences containing diverse modifications.

Enhanced Ribozyme-Catalyzed Recombination and Oligonucleotide Assembly in Peptide-RNA Condensates.

The ability of RNA to catalyze RNA ligation is critical to its central role in many prebiotic model scenarios, in particular the copying of information during self-replication. Prebiotically plausible ribozymes formed from short oligonucleotides can catalyze reversible RNA cleavage and ligation reactions, but harsh conditions or unusual scenarios are often required to promote folding and drive the reaction equilibrium towards ligation. Here, we demonstrate that ribozyme activity is greatly enhanced by charge-mediated phase separation with poly-L-lysine, which shifts the reaction equilibrium from cleavage in solution to ligation in peptide-RNA coaggregates and coacervates. This compartmentalization enables robust isothermal RNA assembly over a broad range of conditions, which can be leveraged to assemble long and complex RNAs from short fragments under mild conditions in the absence of exogenous activation chemistry, bridging the gap between pools of short oligomers and functional RNAs.

Triplex-forming properties and enzymatic incorporation of a base-modified nucleotide capable of duplex DNA recognition at neutral pH.

The sequence-specific recognition of duplex DNA by unmodified parallel triplex-forming oligonucleotides is restricted to low pH conditions due to a necessity for cytosine protonation in the third strand. This has severely restricted their use as gene-targeting agents, as well as for the detection and/or functionalisation of synthetic or genomic DNA. Here I report that the nucleobase 6-amino-5-nitropyridin-2-one (Z) finally overcomes this constraint by acting as an uncharged mimic of protonated cytosine. Synthetic TFOs containing the nucleobase enabled stable and selective triplex formation at oligopurine-oligopyrimidine sequences containing multiple isolated or contiguous GC base pairs at neutral pH and above. Moreover, I demonstrate a universal strategy for the enzymatic assembly of Z-containing TFOs using its commercially available deoxyribonucleotide triphosphate. These findings seek to improve not only the recognition properties of TFOs but also the cost and/or expertise associated with their chemical syntheses.

Synthesis of DNA Origami Scaffolds: Current and Emerging Strategies.

DNA origami nanocarriers have emerged as a promising tool for many biomedical applications, such as biosensing, targeted drug delivery, and cancer immunotherapy. These highly programmable nanoarchitectures are assembled into any shape or size with nanoscale precision by folding a single-stranded DNA scaffold with short complementary oligonucleotides. The standard scaffold strand used to fold DNA origami nanocarriers is usually the M13mp18 bacteriophage's circular single-stranded DNA genome with limited design flexibility in terms of the sequence and size of the final objects. However, with the recent progress in automated DNA origami design-allowing for increasing structural complexity-and the growing number of applications, the need for scalable methods to produce custom scaffolds has become crucial to overcome the limitations of traditional methods for scaffold production. Improved scaffold synthesis strategies will help to broaden the use of DNA origami for more biomedical applications. To this end, several techniques have been developed in recent years for the scalable synthesis of single stranded DNA scaffolds with custom lengths and sequences. This review focuses on these methods and the progress that has been made to address the challenges confronting custom scaffold production for large-scale DNA origami assembly.

Enzymatic synthesis of biphenyl-DNA oligonucleotides.

The incorporation of nucleotides equipped with C-glycosidic aromatic nucleobases into DNA and RNA is an alluring strategy for a number of practical applications including fluorescent labelling of oligonucleotides, expansion of the genetic alphabet for the generation of aptamers and semi-synthetic organisms, or the modulation of excess electron transfer within DNA. However, the generation of C-nucleoside containing oligonucleotides relies mainly on solid-phase synthesis which is quite labor intensive and restricted to short sequences. Here, we explore the possibility of constructing biphenyl-modified DNA sequences using enzymatic synthesis. The presence of multiple biphenyl-units or biphenyl residues modified with electron donors and acceptors permits the incorporation of a single dBphMP nucleotide. Moreover, templates with multiple abasic sites enable the incorporation of up to two dBphMP nucleotides, while TdT-mediated tailing reactions produce single-stranded DNA oligonucleotides with four biphenyl residues appended at the 3'-end.

Oligonucleotides: Current Trends and Innovative Applications in the Synthesis, Characterization, and Purification.

Oligonucleotides (ONs) are gaining increasing importance as a promising novel class of biopharmaceuticals. Thanks to their fundamental role in gene regulation, they can be used to develop custom-made drugs (also called N-to-1) able to act on the gene expression at pre-translational level. With recent approvals of ON-based therapeutics by the Food and Drug Administration (FDA), a growing demand for high-quality chemically modified ONs is emerging and their market is expected to impressively prosper in the near future. To satisfy this growing market demand, a scalable and economically sustainable ON production is needed. In this paper, the state of the art of the whole ON production process is illustrated with the aim of highlighting the most promising routes toward the auspicated market-size production. In particular, the most recent advancements in both the upstream stage, mainly based on solid-phase synthesis and recombinant technology, and the downstream one, focusing on chromatographic techniques, are reviewed. Since ON production is projected to expand to the large scale, automatized multicolumn countercurrent technologies will reasonably be required soon to replace the current ones based on batch single-column operations. This consideration is supported by a recent cutting-edge application of continuous chromatography for the ON purification.

Enzymatic Synthesis of Designer DNA Using Cyclic Reversible Termination and a Universal Template.

Phosphoramidite chemistry remains the industry standard for DNA synthesis despite significant limitations on the length and yield of the oligonucleotide, time restrictions, and hazardous waste production. Herein, we demonstrate the synthesis of single-stranded oligos on a solid surface by DNA polymerases and reverse transcriptases. We report the extension of surface-bound oligonucleotides enabled by transient hybridization of as few as two bases to a neighboring strand. When multiple hybridization structures are possible, each templating a different base, a DNA polymerase or reverse transcriptase can extend the oligonucleotide with any of the complementary bases. Therefore, the sequence of the newly synthesized fragment can be controlled by adding only the desired base as a substrate to the reaction solution. We used this enzymatic approach to synthesize a 20 base oligonucleotide by incorporating reversible terminator dNTPs through a two-step cyclic reversible termination process with a corrected stepwise efficiency over 98%. In our approach, a nascent DNA strand that serves as both primer and template is extended through polymerase-controlled sequential addition of 3'-reversibly blocked nucleotides followed by subsequent cleavage of the 3'-capping group. This process enables oligonucleotide synthesis in an environment not permitted by traditional phosphoramidite methods, eliminates the need for hazardous chemicals, has the potential to provide faster and higher yield results, and synthesizes DNA on a solid support with a free 3' end.

DNA polymerase activity on synthetic N3'→P5' phosphoramidate DNA templates.

Genetic polymers that could plausibly govern life in the universe might inhabit a broad swath of chemical space. A subset of these genetic systems can exchange information with RNA and DNA and could therefore form the basis for model protocells in the laboratory. N3'→P5' phosphoramidate (NP) DNA is defined by a conservative linkage substitution and has shown promise as a protocellular genetic material, but much remains unknown about its functionality and fidelity due to limited enzymatic tools. Conveniently, we find widespread NP-DNA-dependent DNA polymerase activity among reverse transcriptases, an observation consistent with structural studies of the RNA-like conformation of NP-DNA duplexes. Here, we analyze the consequences of this unnatural template linkage on the kinetics and fidelity of DNA polymerization activity catalyzed by wild-type and variant reverse transcriptases. Template-associated deficits in kinetics and fidelity suggest that even highly conservative template modifications give rise to error-prone DNA polymerase activity. Enzymatic copying of NP-DNA sequences is nevertheless an important step toward the future study and engineering of this synthetic genetic polymer.

Enzymatic Synthesis of Backbone-Modified Oligonucleotides Using T4 DNA Ligase.

T4 DNA ligase in high concentrations of certain crowding agents and cosolutes catalyzes the synthesis of a series of backbone-modified oligonucleotides that are difficult to obtain chemically. Backbone-modified nucleic acids are often enzymatically and chemically more stable, making them interesting as potential diagnostic or therapeutic agents, as a biosafety tool, or in nanotechnology. In this article, we describe a small-scale experiment to probe the efficiency of the ligation reaction of modified oligonucleotides in the presence of 3 M betaine and 10% PEG 8000, followed by large-scale ligation with subsequent isolation of the ligated oligonucleotide. The correct product formation can be verified using denaturing polyacrylamide gel electrophoresis and mass spectrometry. © 2019 by John Wiley & Sons, Inc.

Efficiency and fidelity of T3 DNA ligase in ligase-catalysed oligonucleotide polymerisations.

Ligase-catalyzed oligonucleotide polymerisations (LOOPER) can readily generate libraries of diversely-modified nucleic acid polymers, which can be subjected to iterative rounds of in vitro selection to evolve functional activity. While there exist several different DNA ligases, T4 DNA ligase has most often been used for the process. Recently, T3 DNA ligase was shown to be effective in LOOPER; however, little is known about the fidelity and efficiency of this enzyme in LOOPER. In this paper we evaluate the efficiency of T3 DNA ligase and T4 DNA ligase for various codon lengths and compositions within the context of polymerisation fidelity and yield. We find that T3 DNA ligase exhibits high efficiency and fidelity with short codon lengths, but struggles with longer and more complex codon libraries, while T4 DNA ligase exhibits the opposite trend. Interestingly, T3 DNA ligase is unable to accommodate modifications at the 8-position of adenosine when integrated into short codons, which will create challenges in expanding the available codon set for the process. The limitations and strengths of the two ligases are further discussed within the context of LOOPER.

De novo DNA synthesis using polymerase-nucleotide conjugates.

Oligonucleotides are almost exclusively synthesized using the nucleoside phosphoramidite method, even though it is limited to the direct synthesis of ∼200 mers and produces hazardous waste. Here, we describe an oligonucleotide synthesis strategy that uses the template-independent polymerase terminal deoxynucleotidyl transferase (TdT). Each TdT molecule is conjugated to a single deoxyribonucleoside triphosphate (dNTP) molecule that it can incorporate into a primer. After incorporation of the tethered dNTP, the 3' end of the primer remains covalently bound to TdT and is inaccessible to other TdT-dNTP molecules. Cleaving the linkage between TdT and the incorporated nucleotide releases the primer and allows subsequent extension. We demonstrate that TdT-dNTP conjugates can quantitatively extend a primer by a single nucleotide in 10-20 s, and that the scheme can be iterated to write a defined sequence. This approach may form the basis of an enzymatic oligonucleotide synthesizer.

Enzymatic amplification of oligonucleotides in paper substrates.

Several solution-based methods have recently been adapted for use in paper substrates for enzymatic amplification to increase the number of copies of DNA sequences. There is limited information available about the impact of a paper matrix on DNA amplification by enzymatic processes, and about how to optimize conditions to maximize yields. The work reported herein provides insights about the impact of physicochemical properties of a paper matrix, using nuclease-assisted amplification by exonuclease III and nicking endonuclease Nt. Bbv, and a quantum dot (QD) - based Forster Resonance Energy Transfer (FRET) assay to monitor the extent of amplification. The influence of several properties of paper on amplification efficiency and kinetics were investigated, such as surface adsorption of reactants, and pore size. Additional factors that impact amplification processes such as target length and the packing density of oligonucleotide probes on the nanoparticle surfaces were also studied. The work provides guidance for development of more efficient enzymatic target-recycling DNA amplification methods in paper substrates.

Ammonium and arsenic trioxide are potent facilitators of oligonucleotide function when delivered by gymnosis.

Oligonucleotide (ON) concentrations employed for therapeutic applications vary widely, but in general are high enough to raise significant concerns for off target effects and cellular toxicity. However, lowering ON concentrations reduces the chances of a therapeutic response, since typically relatively small amounts of ON are taken up by targeted cells in tissue culture. It is therefore imperative to identify new strategies to improve the concentration dependence of ON function. In this work, we have identified ammonium ion (NH4+) as a non-toxic potent enhancer of ON activity in the nucleus and cytoplasm following delivery by gymnosis. NH4+ is a metabolite that has been extensively employed as diuretic, expectorant, for the treatment of renal calculi and in a variety of other diseases. Enhancement of function can be found in attached and suspension cells, including in difficult-to-transfect Jurkat T and CEM T cells. We have also demonstrated that NH4+ can synergistically interact with arsenic trioxide (arsenite) to further promote ON function without producing any apparent increased cellular toxicity. These small, inexpensive, widely distributed molecules could be useful not only in laboratory experiments but potentially in therapeutic ON-based combinatorial strategy for clinical applications.

Carborane-linked 2'-deoxyuridine 5'-O-triphosphate as building block for polymerase synthesis of carborane-modified DNA.

5-[(p-Carborane-2-yl)ethynyl]-2'-deoxyuridine 5'-O-triphosphate was synthesized and used as a good substrate in enzymatic construction of carborane-modified DNA or oligonucleotides containing up to 21 carborane moieties in primer extension reactions by DNA polymerases.

Fermentation properties of isomaltooligosaccharides are affected by human fecal enterotypes.

Isomaltooligosaccharides (IMOs) are enzymatically synthesized oligosaccharides that have potential prebiotic effects. Five IMO substrates with 2-16° of polymerization (DP) were studied for their fermentation capacities using human microbiomes in an in vitro batch fermentation model. Eleven fecal slurries belonging to three enterotypes, including the Bacteroides-, Prevotella- and Mixed-type, exhibited different degradation rates for long chain IMOs (DP 7 to 16). In contrast, the degradation rates for short chain IMOs (DP 2 to 6) were not affected by enterotypes. Both 16S rRNA gene sequencing and quantitative PCR demonstrated that, after fermentation, the Bifidobacterium growth with IMOs was primarily detected in the Bacteroides- and Mixed-type (non-Prevotella-type), and to a lesser degree in the Prevotella-type. Interestingly, the Prevotella-type microbiome had higher levels of propionic acid and butyric acid production than non-Prevotella-type microbiome after IMOs fermentation. Moreover, principal coordinate analysis (PCoA) of both denaturing gradient gel electrophoresis (DGGE) profiling and 16S rRNA sequencing data demonstrated that the microbiome community compositions were separately clustered based on IMO chain length, suggesting significant impact of DP on the bacterial community structure. The current results clearly demonstrated that the IMO chain length could modulate the structure and composition of the human colonic microbiome. Different responses to short and long chain IMOs were observed from three human enterotypes, indicating that IMOs may be used as therapeutic substrates for directly altering human colonic bacteria.

Enzymatic Synthesis of 7',5'-Bicyclo-DNA Oligonucleotides.

The selection of artificial genetic polymers with tailor-made properties for their application in synthetic biology requires the exploration of new nucleosidic scaffolds that can be used in selection experiments. Herein, we describe the synthesis of a bicyclo-DNA triphosphate (i.e., 7',5'-bc-TTP) and show its potential to serve for the generation of new xenonucleic acids (XNAs) based on this scaffold. 7',5'-bc-TTP is a good substrate for Therminator DNA polymerase, and up to seven modified units can be incorporated into a growing DNA chain. In addition, this scaffold sustains XNA-dependent DNA synthesis and potentially also XNA-dependent XNA synthesis. However, DNA-dependent XNA synthesis on longer templates is hampered by competitive misincorporation of deoxyadenosine triphosphate (dATP) caused by the slow rate of incorporation of 7',5'-bc-TTP.

Structure-activity relationships of the ATP cofactor in ligase-catalysed oligonucleotide polymerisations.

A T4 DNA ligase-catalysed oligonucleotide polymerisation process has been recently developed to enable the incorporation of multiple functional groups throughout a nucleic acid polymer. T4 DNA ligase requires ATP as a cofactor to catalyse phosphodiester bond formation during the polymerisation process. Herein, we describe the structure-activity relationship of ATP within the context of T4 DNA ligase-catalyzed oligonucleotide polymerisation. Using high-throughput sequencing, we study not only the influence of ATP modification on polymerisation efficiency, but also on the fidelity and sequence bias of the polymerisation process.

Synthesis of chemically modified DNA.

Naturally occurring DNA is encoded by the four nucleobases adenine, cytosine, guanine and thymine. Yet minor chemical modifications to these bases, such as methylation, can significantly alter DNA function, and more drastic changes, such as replacement with unnatural base pairs, could expand its function. In order to realize the full potential of DNA in therapeutic and synthetic biology applications, our ability to 'write' long modified DNA in a controlled manner must be improved. This review highlights methods currently used for the synthesis of moderately long chemically modified nucleic acids (up to 1000 bp), their limitations and areas for future expansion.

A cyclopropene-modified nucleotide for site-specific RNA labeling using genetic alphabet expansion transcription.

Site-specific RNA modification with methyl cyclopropene moieties is performed by T7 in vitro transcription. An existing unnatural base is functionalized with a cyclopropene moiety and used in transcription reactions to produce site-specifically cyclopropene-modified RNA molecules. The posttranscriptional inverse electron demand Diels-Alder cycloaddition reaction with a selected tetrazine-fluorophore conjugate is demonstrated.