Kinetic analysis of N-alkylaryl carboxamide hexitol nucleotides as substrates for evolved polymerases.

Six 1',5'-anhydrohexitol uridine triphosphates were synthesized with aromatic substitutions appended via a carboxamide linker to the 5-position of their bases. An improved method for obtaining such 5-substituted hexitol nucleosides and nucleotides is described. The incorporation profile of the nucleotide analogues into a DNA duplex overhang using recently evolved XNA polymerases is compared. Long, mixed HNA sequences featuring the base modifications are generated. The apparent binding affinity of four of the nucleotides to the enzyme, the rate of the chemical step and of product release, plus the specificity constant for the incorporation of these modified nucleotides into a DNA duplex overhang using the HNA polymerase T6G12_I521L are determined via pre-steady-state kinetics. HNA polymers displaying aromatic functional groups could have significant impact on the isolation of stable and high-affinity binders and catalysts, or on the design of nanomaterials.

Bacterial Cell Display as a Robust and Versatile Platform for Engineering Low-Affinity Ligands and Enzymes.

Directed evolution has been remarkably successful at expanding the chemical and functional boundaries of biology. That progress is heavily dependent on the robustness and flexibility of the available selection platforms, given the significant cost to (re)develop a given platform to target a new desired function. Bacterial cell display has a significant track record as a viable strategy for the engineering of mesophilic enzymes, as enzyme activity can be probed directly and free from interference from the cellular milieu, but its adoption has lagged behind other display-based methods. Herein, we report the development of SNAP as a quantitative reporter for bacterial cell display, which enables fast troubleshooting and the systematic development of the display-based selection platform, thus improving its robustness. In addition, we demonstrate that even weak interactions between displayed proteins and nucleic acids can be harnessed for the specific labelling of bacterial cells, allowing functional characterisation of DNA binding proteins and enzymes, thus making it a highly flexible platform for these biochemical functions. Together, this establishes bacterial display as a robust and flexible platform, ideally suited for the systematic engineering of ligands and enzymes needed for XNA molecular biology.

Whole cell-SELEX of aptamers with a tyrosine-like side chain against live bacteria.

In an effort to expand the binding and recognition capabilities of aptamers, a nucleoside triphosphate modified with a phenol that mimics the side chain of tyrosine was used in the selection of DNA aptamers against live bacteria. Of multiple modified aptamers that were isolated against Escherichia coli DH5α cells, one aptamer displays high selectivity and affinity for the target cells and is greatly enriched for phenol-modified dU nucleotides (dUy, 47.5%). When the same sequences are synthesized with TTP, no binding is observed. Taken together, these findings highlight the value of using modified nucleotide triphosphates in aptamer selections and portends success in SELEX against an array of whole cells as targets.

Nucleosides with Transposed Base or 4'-Hydroxymethyl Moieties and Their Corresponding Oligonucleotides.

This review focuses on 4'-hydroxymethyl- or nucleobase-transposed nucleosides, nucleotides, and nucleoside phosphonates, their stereoisomers, and their close analogues. The biological activities of all known 4'-hydroxymethyl- or nucleobase-transposed nucleosides, nucleotides, and nucleoside phosphonates as potential antiviral or anticancer agents are compiled. The routes that have been taken for the chemical synthesis of such nucleoside derivatives are described, with special attention to the innovative strategies. The enzymatic synthesis, base-pairing properties, structure, and stability of oligonucleotides containing nucleobase- or 4'-hydroxymethyl-transposed nucleotides are discussed. The use of oligonucleotides containing nucleobase- or 4'-hydroxymethyl-transposed nucleotides as small oligonucleotide (e.g., human immunodeficiency virus integrase) inhibitors, in applications such as antisense therapy, silencing RNA (siRNA), or aptamer selections, is detailed.

Catalysing mirror life.

Origin of life: Although current life is homochiral (with D nucleic acids), little is known about how homochirality emerged or even if it was a necessary step. The isolation of cross-chiral nucleic acid ligases demonstrate that an early heterochiral life could have been possible.

NICTABA and UDA, two GlcNAc-binding lectins with unique antiviral activity profiles.

OBJECTIVES: This study aimed to assess the antiviral properties of a unique lectin (NICTABA) produced by the tobacco plant, Nicotiana tabacum. METHODS: Cellular assays were used to investigate the antiviral activity of NICTABA and Urtica dioica agglutinin (UDA). Surface plasmon resonance (SPR) studies were performed to study the sugar specificity and the interactions of both lectins with the envelope glycoproteins of HIV-1. RESULTS: The N-acetyl-d-glucosamine (GlcNAc)-binding lectins exhibited broad-spectrum activity against several families of enveloped viruses including influenza A/B, Dengue virus type 2, herpes simplex virus types 1 and 2 and HIV-1/2. The IC50 of NICTABA for various HIV-1 strains, clinical isolates and HIV-2 assessed in PBMCs ranged from 5 to 30 nM. Furthermore, NICTABA inhibited syncytium formation between persistently HIV-1-infected T cells and uninfected CD4+ T lymphocytes and prevented DC-SIGN-mediated HIV-1 transmission to CD4+ target T lymphocytes. However, unlike many other antiviral carbohydrate-binding agents (CBAs) described so far, NICTABA did not block HIV-1 capture to DC-SIGN+ cells and it did not interfere with the binding of the human monoclonal antibody 2G12 to gp120. SPR studies with HIV-1 envelope glycoproteins showed that the affinity of NICTABA for gp120 and gp41 was in the low nanomolar range. The specific binding of NICTABA to gp120 could be prevented in the presence of a GlcNAc trimer, but not in the presence of mannose trimers. NICTABA displayed no antiviral activity against non-enveloped viruses. CONCLUSIONS: Since CBAs possess a high genetic barrier for the development of viral resistance and NICTABA shows a broad antiviral activity profile, this CBA may qualify as a potential antiviral candidate with a pleiotropic mode of action aimed at targeting the entry of enveloped viruses.

A novel framework for engineering protein loops exploring length and compositional variation.

Insertions and deletions (indels) are known to affect function, biophysical properties and substrate specificity of enzymes, and they play a central role in evolution. Despite such clear significance, this class of mutation remains an underexploited tool in protein engineering with few available platforms capable of systematically generating and analysing libraries of varying sequence composition and length. We present a novel DNA assembly platform (InDel assembly), based on cycles of endonuclease restriction digestion and ligation of standardised dsDNA building blocks, that can generate libraries exploring both composition and sequence length variation. In addition, we developed a framework to analyse the output of selection from InDel-generated libraries, combining next generation sequencing and alignment-free strategies for sequence analysis. We demonstrate the approach by engineering the well-characterized TEM-1 ß-lactamase Ω-loop, involved in substrate specificity, identifying multiple novel extended spectrum ß-lactamases with loops of modified length and composition-areas of the sequence space not previously explored. Together, the InDel assembly and analysis platforms provide an efficient route to engineer protein loops or linkers where sequence length and composition are both essential functional parameters.

Pradimicin S, a highly soluble nonpeptidic small-size carbohydrate-binding antibiotic, is an anti-HIV drug lead for both microbicidal and systemic use.

Pradimicin S (PRM-S) is a highly water-soluble, negatively charged derivative of the antibiotic pradimicin A (PRM-A) in which the terminal xylose moiety has been replaced by 3-sulfated glucose. PRM-S does not prevent human immunodeficiency virus (HIV) adsorption on CD4(+) T cells, but it blocks virus entry into its target cells. It inhibits a wide variety of HIV-1 laboratory strains and clinical isolates, HIV-2, and simian immunodeficiency virus (SIV) in various cell culture systems (50% and 90% effective concentrations [EC(50)s and EC(90)s] invariably in the lower micromolar range). PRM-S inhibits syncytium formation between persistently HIV-1- and SIV-infected cells and uninfected CD4(+) T lymphocytes, and prevents dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN)-mediated HIV-1 and SIV capture and subsequent virus transmission to CD4(+) T cells. Surface plasmon resonance (SPR) studies revealed that PRM-S strongly binds to gp120 in a Ca(2+)-dependent manner at an affinity constant (K(D)) in the higher nanomolar range. Its anti-HIV activity and HIV-1 gp120-binding properties can be dose-dependently reversed in the presence of an (alpha-1,2)mannose trimer. Dose-escalating exposure of HIV-1-infected cells to PRM-S eventually led to the isolation of mutant virus strains that had various deleted N-glycosylation sites in the envelope gp120 with a strong preference for the deletion of the high-mannose-type glycans. Genotypic resistance development occurred slowly, and significant phenotypic resistance occurred only after the sequential appearance of up to six mutations in gp120, pointing to a high genetic barrier of PRM-S. The antibiotic is nontoxic against a variety of cell lines, is not mitogenic, and does not induce cytokines and chemokines in peripheral blood mononuclear cells as determined by the Bio-Plex human cytokine 27-plex assay. It proved stable at high temperature and low pH. Therefore, PRM-S may qualify as a potential anti-HIV drug candidate for further (pre)clinical studies, including its microbicidal use.

Polymerase-catalysed incorporation of glucose nucleotides into a DNA duplex.

The enzymatic recognition of six-membered ring nucleoside triphosphates--in particular the 6'-triphosphates of (beta-D-glucopyranosyl)thymine, (2',3'-dideoxy-beta-D-glucopyranosyl)thymine, (3',4'-dideoxy-beta-D-glucopyranosyl)thymine and (2',3'-dideoxy-beta-D-glucopyranosyl)adenine--was investigated. Despite the facts that the pyranose nucleic acids obtained by polymerisation of these monomers do not hybridise in solution with DNA and that the geometry of a DNA strand in a natural duplex differs from that of a pyranose nucleic acid, elongation of the DNA duplex with all four nucleotide analogues by Vent (exo(-)) polymerase was observed. Modelling experiments showed that hydrogen bonds are formed when 2',3'-dideoxy-beta-homo-T building blocks or beta-D-gluco-T building blocks are incorporated opposite adenosine residues in the template but not when they are incorporated opposite thymine residues in the template. The model shows a near perfect alignment of a secondary hydroxy group at the end of the primer and the alpha-phosphate group of the incoming triphosphate. The results of these experiments provide new information on the role of the active site of the enzyme in the polymerisation reaction.

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.

Full Pre-Steady-State Kinetic Analysis of Single Nucleotide Incorporation by DNA Polymerases.

By measuring a DNA polymerase incorporation reaction on a very short time scale (5 ms to 10 s) and with a high enzyme concentration, the isolated event of a single nucleotide incorporation can be analyzed. Practically, this is done using a quench-flow instrument, which allows the rapid mixing of the enzyme-primer/template with the nucleotide substrate, after which the reaction is quenched and analyzed. We describe how to titrate the enzyme active site, how to determine, via a scouting experiment, the rate-limiting step in the polymerization reaction, and how to measure the apparent kpol , Kd(DNA) , and Kd(N) using burst or single-turnover experiments. We include equations for the calculation of the processivity of the polymerase, its nucleotide incorporation specificity and preference, and the activation energy difference for the incorporation of an incorrect nucleotide. Data analysis is discussed, as this is an essential part of accurate data generation in kinetic analyses. © 2019 by John Wiley & Sons, Inc.

Enzymatic polymerization of phosphonate nucleosides.

5'-O-phosphonomethyl-2'-deoxyadenosine (PMdA) proved to be a good substrate of the Therminator polymerase. In this article, we investigated whether the A, C, T and U analogues of this phosphonate nucleoside (PMdN) series can function as substrates of natural DNA polymerases. PMdT and PMdU could only be polymerized enzymatically to a limited extent. Nevertheless, PMdA and PMdC could be incorporated into a DNA duplex with complete chain elongation by all the DNA polymerases tested. A mixed sequence of four nucleotides containing modified C, T and A residues could be obtained with the Vent(exo(-)) and Therminator polymerases. The kinetic values for the incorporation of PMdA by Vent(exo(-)) polymerase were determined; a reduced K(M) value was found for the incorporation of PMdA compared to the natural substrate. Future polymerase directed evolution studies will allow us to select an enzyme with a heightened capacity to process these modified DNA building blocks into modified strands.

XNA ligation using T4 DNA ligase in crowding conditions.

T4 DNA ligase is capable of ligating 2'OMe-RNA duplexes, HNA, LNA and FANA mixed sequences in the presence of 10% w/v PEG8000 and 3 M betaine. The enzymatic joining of oligonucleotides containing multiple consecutive XNA nucleotides at the ligation site has not been reported before.

Investigation of the DNA-dependent cyclohexenyl nucleic acid polymerization and the cyclohexenyl nucleic acid-dependent DNA polymerization.

DNA polymerases from different evolutionary families [Vent (exo-) DNA polymerase from the B-family polymerases, Taq DNA polymerase from the A-family polymerases and HIV reverse transcriptase from the reverse transcriptase family] were examined for their ability to incorporate the sugar-modified cyclohexenyl nucleoside triphosphates. All enzymes were able to use the cyclohexenyl nucleotides as a substrate. Using Vent (exo-) DNA polymerase and HIV reverse transcriptase, we were even able to incorporate seven consecutive cyclohexenyl nucleotides. Using a cyclohexenyl nucleic acid (CeNA) template, all enzymes tested were also able to synthesize a short DNA fragment. Since the DNA-dependent CeNA polymerization and the CeNA-dependent DNA polymerization is possible to a limited extend, we suggest CeNA as an ideal candidate to use in directed evolution methods for the development of a polymerase capable of replicating CeNA.

A method for selecting modified DNAzymes without the use of modified DNA as a template in PCR.

Modified DNAzyme selections typically depend on recopying catalytically active modified DNA (mDNA) into cDNA in a PCR amplification step. However mDNA is often a poor template in PCR. Herein we propose a selection method in which the catalytically active, mDNA strand is covalently linked to the unmodified DNA template strand from which it was polymerized. Following selection, the unmodified DNA template is amplified in a PCR instead of the mDNA. This method circumvents the PCR amplification of mDNA.

Synthetic genetic polymers capable of heredity and evolution.

Genetic information storage and processing rely on just two polymers, DNA and RNA, yet whether their role reflects evolutionary history or fundamental functional constraints is currently unknown. With the use of polymerase evolution and design, we show that genetic information can be stored in and recovered from six alternative genetic polymers based on simple nucleic acid architectures not found in nature [xeno-nucleic acids (XNAs)]. We also select XNA aptamers, which bind their targets with high affinity and specificity, demonstrating that beyond heredity, specific XNAs have the capacity for Darwinian evolution and folding into defined structures. Thus, heredity and evolution, two hallmarks of life, are not limited to DNA and RNA but are likely to be emergent properties of polymers capable of information storage.

Targeting HIV entry through interaction with envelope glycoprotein 120 (gp120): synthesis and antiviral evaluation of 1,3,5-triazines with aromatic amino acids.

On the basis of the interesting inhibitory properties that lectins show against HIV-replication through their interaction with glycoprotein 120 (gp120), we here describe the design, synthesis, and anti-HIV evaluation of three series of 1,3,5-triazine derivatives (monomers, dimers, and trimers) functionalized with aromatic amino acids meant to mimic interactions that lectins establish with gp120. While monomers were inactive against HIV replication, dimers showed limited anti-HIV activity that is, however, considerably more significant in the trimers series, with EC(50) values in the lower µM range. These findings most likely reflect the requirement of multivalency of the 1,3,5-triazine derivatives to display anti-HIV activity, as lectins do. The pronounced anti-HIV activity (EC(50) ∼ 20 µM) is accompanied by the absence of toxicity in CEM T-cell line (CC(50) > 250 µM). Moreover, SPR experiments revealed that the prototype trimers with a central core of 2,4,6-triethylbenzene and six l-Trp or six l-Tyr residues at the periphery were efficient binders of CXCR4- and CCR5-tropic HIV-1 gp120 (estimated K(D): lower micromolar range). The collected data support the interest of this novel family of anti-HIV agents and qualify them as potential novel microbicide lead compounds.

Synthesis of 2'-N-formamido nucleosides and biological evaluation.

The 2'-N-formamide derivatives of adenosine, cytidine, and 9-beta-d-arabinofuranosyladenine were synthesized and tested (as triphosphate) for their substrate capacities for the HCV NS5B polymerase.