Rapid and efficient synthesis of nucleoside 5'-0-(1-thiotriphosphates), 5'-triphosphates and 2',3'-cyclophosphorothioates using 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one

Negria S., Jia Y., Setterholm N.A., Barpuzary B., Chaput J.C.

Saito-Tarashima N., Koma T., Hinotani N., Yoshida K., Ogasa M., Murai A., Inoue S., Kondo T., Doi N., Tsuneyama K., Nomaguchi M., Minakawa N.

Fehlau M., Westarp S., Neubauer P., Kurreck A.

Nucleoside-5′-triphosphates (5′-NTPs) are essential building blocks of nucleic acids in nature and play an important role in molecular biology, diagnostics, and mRNA therapeutic synthesis. Chemical synthesis has long been the standard method for producing modified 5′-NTPs. However, chemical routes face limitations, including low regio- and stereoselectivity, along with the need for protection/deprotection cycles, resulting in low yields, high costs, and lengthy processes. In contrast, enzymatic synthesis methods offer significant advantages, such as improved regio- and stereoselectivity and the use of mild reaction conditions, which often leads to higher product yields in “one-pot” reactions. Despite the extensive review of chemical synthesis routes for 5′-NTPs, there has not yet been any comprehensive analysis of enzymatic approaches. Initially, this review provides a brief overview of the enzymes involved in nucleotide metabolism, introducing valuable biocatalysts for 5’-NTP synthesis. Furthermore, the available enzymatic methods for efficient 5′-NTP synthesis using purified enzymes and starting from either nucleobases or nucleosides are examined, highlighting their respective advantages and disadvantages. Special attention is also given to the importance of ATP regeneration systems for 5′-NTP synthesis. We aim to demonstrate the remarkable potential of enzymatic in vitro cascade reactions, promoting their broader application in both basic research and industry.

Kokoris M., McRuer R., Nabavi M., Jacobs A., Prindle M., Cech C., Berg K., Lehmann T., Machacek C., Tabone J., Chandrasekar J., McGee L., Lopez M., Reid T., Williams C., et. al.

AbstractRemarkable advances in high-throughput sequencing have enabled major biological discoveries and clinical applications, but achieving wider distribution and use depends critically on further improvements in scale and cost reduction. Nanopore sequencing has long held the promise for such progress, but has had limited market penetration. This is because efficient and accurate nanopore sequencing of nucleic acids has been challenged by fundamental signal-to-noise limitations resulting from the poor spatial resolution and molecular distinction of nucleobases. Here, we describe Sequencing by Expansion (SBX), a single-molecule sequencing technology that overcomes these limitations by using a biochemical conversion process to encode the sequence of a target nucleic acid molecule into an Xpandomer, a highly measurable surrogate polymer. Expanding over 50 times longer than the parent DNA templates, Xpandomers are engineered with high signal-to-noise reporter codes to enable facile, high-accuracy nanopore sequencing. We demonstrate the performance of SBX and present the specialized molecular structures, chemistries, enzymes and methods that enable it. The innovative molecular and systems engineering in SBX create a transformative technology to address the needs of existing and emerging sequencing applications.

Tang B., Wang P., Shen Y.

A new cleavable azo linker was synthesized and reacted with 5-iodo-2 '-deoxyuridine, followed by triphosphorylation, and finally labeled with Cy3 to give the desired product dUTP-Azo linker -Cy3 as potential...

Han X., Sczepanski J.T.

This work describes a straightforward strategy for assembling long l-RNAs via the joining of two or more shorter fragments using cross-chiral ligase ribozymes together with new substrate activation chemistry.

Tang B., Wang P., Shen Y.

Marcos Anghinoni J., Irum, Ur Rashid H., João Lenardão E., Santos Silva M.

Abstract31P NMR spectroscopy is a consolidated tool for the characterization of organophosphorus compounds and, more recently, for reaction monitoring. The evolution of organic synthesis, mainly due to the combination of elaborated building blocks with enabling technologies, generated great challenges to understand and to optimize the synthetic methodologies. In this sense, 31P NMR experiments also became a routine technique for reaction monitoring, accessing products and side products yields, chiral recognition, kinetic data, intermediates, as well as basic organic parameters, such as acid‐base and hydrogen‐bonding. This review deals with these aspects demonstrating the essential role of the 31P NMR spectroscopy. The recent publications (the last ten years) will be explored, discussing the experiments of 31P NMR and the strategies accomplished to detect and/or quantify distinct organophosphorus molecules, approaching reaction mechanism, stability, stereochemistry, and the utility as a probe.

Cochrane W.G., Bare G.A., Joyce G.F., Horning D.P.

An RNA ligase ribozyme that catalyzes the joining of RNA molecules of the opposite chiral handedness was optimized for the ability to synthesize its own enantiomer from two component fragments. The mirror-image D- and L-ligases operate in concert to provide a system for cross-chiral replication, whereby they catalyze each other’s synthesis and undergo mutual amplification at constant temperature, with apparent exponential growth and a doubling time of about 1 h. Neither the D- nor the L-RNA components alone can achieve autocatalytic self-replication. Cross-chiral exponential amplification can be continued indefinitely through a serial-transfer process that provides an ongoing supply of the component D- and L-substrates. Unlike the familiar paradigm of semiconservative nucleic acid replication that relies on Watson–Crick pairing between complementary strands, cross-chiral replication relies on tertiary interactions between structured nucleic acids “across the mirror.” There are few examples, outside of biology, of autocatalytic self-replication systems that undergo exponential amplification and there are no prior examples, in either biological or chemical systems, of cross-chiral replication enabling exponential amplification.

Karalkar N.B., Kent T., Tredinnick T., Betancurt-Anzola L., Delarue M., Pomerantz R., Benner S.A.

AbstractA route to prepare ribonucleoside triphosphates featuring a 3’-aminoxy (3’-O-NH2) removable blocking group is reported here. We then show that versions of two DNA polymerases, human DNA polymerase theta (Polθ) and mimiviral PrimPol, accept these triphosphates as substrates to add single nucleotides to an RNA primer under engineered conditions. Cleaving the O-N bond in the 3’-O-NH2group within the extended primer regenerates the 3’-OH group, facilitating subsequent polymerase cycles that add a second, selected, nucleotide. These enzymes and triphosphates together enable template-independent enzymatic RNA synthesis (TIERS) exploiting a cyclic reversible termination framework. The study shows that this process is ready for instrument adaptation by using it to add three ribonucleotides in three cycles using an engineered Polθ. This work creates a new way to synthesize RNA with a de novo defined sequence, without requiring the protecting groups, hazardous solvents, and sensitive reagents that bedevil phosphoramidite-based RNA synthesis.

Han X., Sczepanski J.T.

ABSTRACTDespite the growing interest in mirror-image L-oligonucleotides, both as a robust nucleic acid analogue and as an artificial genetic polymer, their broader adoption in research and medicine remains hindered by challenges associated with the synthesis of long sequences, especially for L-RNA. Herein, we present a novel strategy for assembling long L-RNAs via the joining of two or more shorter fragments using cross-chiral ligase ribozymes together with new substrate activation chemistry. We show that 5′-monophosphorylated L-RNA, which is readily prepared by solid phase synthesis, can be activated by chemical attachment of a 5′-adenosine monophosphate (AMP) or diphosphate (ADP), yielding 5′-adenosyl (di-or tri-) phosphate L-RNA. The activation reaction is performed in mild aqueous conditions, proceeds efficiently with short or large L-RNA, and, yielding few biproducts, requires little or no further purification after activation. Importantly, both groups, when added to L-RNA, are compatible with ribozyme-mediated ligation, with the 5′-adenosyl triphosphate permitting rapid and efficient joining of multiple, long L-RNA strands. This is exemplified by the assembly of a 129-nt L-RNA molecule via a single cross-chiral ligation event. Overall, by relying on ribozymes that can be readily prepared byin vitrotranscription and L-RNA substrates that can be activated through simple chemistry, these methods are expected to make long L-RNAs more accessible to a wider range of researchers and facilitate the expansion of L-ON-based technologies.

Novgorodtseva A.I., Lomzov A.A., Vasilyeva S.V.

This review article is focused on the progress made in the synthesis of 5′-α-P-modified nucleoside triphosphates (α-phosphate mimetics). A variety of α-P-modified nucleoside triphosphates (NTPαXYs, Y = O, S; X = S, Se, BH3, alkyl, amine, N-alkyl, imido, or others) have been developed. There is a unique class of nucleoside triphosphate analogs with different properties. The main chemical approaches to the synthesis of NTPαXYs are analyzed and systematized here. Using the data presented here on the diversity of NTPαXYs and their synthesis protocols, it is possible to select an appropriate method for obtaining a desired α-phosphate mimetic. Triphosphates’ substrate properties toward nucleic acid metabolism enzymes are highlighted too. We reviewed some of the most prominent applications of NTPαXYs including the use of modified dNTPs in studies on mechanisms of action of polymerases or in systematic evolution of ligands by exponential enrichment (SELEX). The presence of heteroatoms such as sulfur, selenium, or boron in α-phosphate makes modified triphosphates nuclease resistant. The most distinctive feature of NTPαXYs is that they can be recognized by polymerases. As a result, S-, Se-, or BH3-modified phosphate residues can be incorporated into DNA or RNA. This property has made NTPαXYs a multifunctional tool in molecular biology. This review will be of interest to synthetic chemists, biochemists, biotechnologists, or biologists engaged in basic or applied research.

Saintomé C., Monfret O., Doisneau G., Guianvarc'h D.

AbstractA variety of proteins interact with DNA and RNA, including polymerases, histones, ribosomes, transcription factors, and repair enzymes. However, the transient non‐covalent nature of these interactions poses challenges for analysis. Introducing a covalent bond between proteins and DNA via photochemical activation of a photosensitive functional group introduced onto nucleic acids offers a means to stabilize these often weak interactions without significantly altering the binding interface. Consequently, photoactivatable oligonucleotides are powerful tools for investigating nucleic acid‐protein interactions involved in numerous biological and pathological processes. In this review, we provide a comprehensive overview of the chemical tools developed so far and the different strategies used for incorporating the most commonly used photoreactive reagents into oligonucleotide probes or nucleic acids. Furthermore, we illustrate their application with several examples including protein binding site mapping, identification of protein binding partners, and in cell studies.

Takezawa Y., Shionoya M.

Metal-mediated artificial base pairs are one of the most promising building blocks for constructing DNA-based supramolecules and functional materials. These base pairs are formed by coordination bonds between ligand-type nucleobases...

Barpuzary B., Negria S., Chaput J.C.

Threofuranosyl nucleic acid (TNA), an artificial genetic polymer known for its nuclease resistance and acid stability, has grown in popularity as a genetically-encoded material for applications in synthetic biology and biomedicine.