Novel insights into the role of translesion synthesis polymerase in DNA incorporation and bypass of 5-fluorouracil in colorectal cancer.

5-Fluorouracil (5-FU) is the first-line chemotherapeutic agent in colorectal cancer, and resistance to 5-FU easily emerges. One of the mechanisms of drug action and resistance of 5-FU is through DNA incorporation. Our quantitative reverse-transcription PCR data showed that one of the translesion synthesis (TLS) DNA polymerases, DNA polymerase η (polη), was upregulated within 72 h upon 5-FU administration at 1 and 10 µM, indicating that polη is one of the first responding polymerases, and the only TLS polymerase, upon the 5-FU treatment to incorporate 5-FU into DNA. Our kinetic studies revealed that 5-fluoro-2'-deoxyuridine triphosphate (5FdUTP) was incorporated across dA 41 and 28 times more efficiently than across dG and across inosine, respectively, by polη indicating that the mutagenicity of 5-FU incorporation is higher in the presence of inosine and that DNA lesions could lead to more mutagenic incorporation of 5-FU. Our polη crystal structures complexed with DNA and 5FdUTP revealed that dA:5FdUTP base pair is like dA:dTTP in the active site of polη, while 5FdUTP adopted 4-enol tautomer in the base pairs with dG and HX increasing the insertion efficiency compared to dG:dTTP for the incorrect insertions. These studies confirm that polη engages in the DNA incorporation and bypass of 5-FU.

Using Cy5-dUTP labelling of RPA-amplicons with downstream microarray analysis for the detection of antibiotic resistance genes.

In this report we describe Cy5-dUTP labelling of recombinase-polymerase-amplification (RPA) products directly during the amplification process for the first time. Nucleic acid amplification techniques, especially polymerase-chain-reaction as well as various isothermal amplification methods such as RPA, becomes a promising tool in the detection of pathogens and target specific genes. Actually, RPA even provides more advantages. This isothermal method got popular in point of care diagnostics because of its speed and sensitivity but requires pre-labelled primer or probes for a following detection of the amplicons. To overcome this disadvantages, we performed an labelling of RPA-amplicons with Cy5-dUTP without the need of pre-labelled primers. The amplification results of various multiple antibiotic resistance genes indicating great potential as a flexible and promising tool with high specific and sensitive detection capabilities of the target genes. After the determination of an appropriate rate of 1% Cy5-dUTP and 99% unlabelled dTTP we were able to detect the blaCTX-M15 gene in less than 1.6E-03 ng genomic DNA corresponding to approximately 200 cfu of Escherichia coli cells in only 40 min amplification time.

Programmable Site-Specific Functionalization of DNA Origami with Polynucleotide Brushes.

Combining surface-initiated, TdT (terminal deoxynucleotidyl transferase) catalyzed enzymatic polymerization (SI-TcEP) with precisely engineered DNA origami nanostructures (DONs) presents an innovative pathway for the generation of stable, polynucleotide brush-functionalized DNA nanostructures. We demonstrate that SI-TcEP can site-specifically pattern DONs with brushes containing both natural and non-natural nucleotides. The brush functionalization can be precisely controlled in terms of the location of initiation sites on the origami core and the brush height and composition. Coarse-grained simulations predict the conformation of the brush-functionalized DONs that agree well with the experimentally observed morphologies. We find that polynucleotide brush-functionalization increases the nuclease resistance of DONs significantly, and that this stability can be spatially programmed through the site-specific growth of polynucleotide brushes. The ability to site-specifically decorate DONs with brushes of natural and non-natural nucleotides provides access to a large range of functionalized DON architectures that would allow for further supramolecular assembly, and for potential applications in smart nanoscale delivery systems.

Rolling circle amplification with fluorescently labeled dUTP-balancing the yield and degree of labeling.

Detection methods based on rolling circle amplification (RCA) have been applied to a large number of targets in molecular biology. The key feature of RCA-based methods as well as other nucleic acid amplification methods is their exceptional sensitivity, which allows the detection of molecules at low concentrations, achieved by signal amplification due to nucleic acid magnification and subsequent detection. Variations on the method, such as immuno-RCA, extend the range of potential targets that can be detected. Employing fluorescently labeled nucleotides for direct incorporation into an amplification product is an attractive method for RCA product detection. However, the effectiveness of this approach remains doubtful. In our study, we utilized different modified dUTPs, including sulfo-cyanine3-dUTP, sulfo-cyanine5-dUTP, sulfo-cyanine5.5-dUTP, BDP-FL-dUTP, and amino-11-dUTP, to investigate whether the properties of the fluorophore used for modification affected the reaction yield and effectiveness of incorporation of nucleotide analogs by phi29 DNA polymerase. Among the modified dUTPs, sulfo-cyanine3-dUTP demonstrated the highest incorporation effectiveness, equal to 4-9 labels per 1000 nucleotides. The mean length of the RCA product was estimated to be approximately 175,000 nucleotides. The total increase in fluorescence from a single target/product complex was 850 times. The results obtained in the study illustrate the possibility of successful application of nucleotide analogs for RCA detection and present quantitative characteristics of fluorescently labeled dUTPs to be incorporated into RCA products.

Advanced Spectroscopy and APBS Modeling for Determination of the Role of His190 and Trp103 in Mouse Thymidylate Synthase Interaction with Selected dUMP Analogues.

A homo-dimeric enzyme, thymidylate synthase (TS), has been a long-standing molecular target in chemotherapy. To further elucidate properties and interactions with ligands of wild-type mouse thymidylate synthase (mTS) and its two single mutants, H190A and W103G, spectroscopic and theoretical investigations have been employed. In these mutants, histidine at position 190 and tryptophan at position 103 are substituted with alanine and glycine, respectively. Several emission-based spectroscopy methods used in the paper demonstrate an especially important role for Trp 103 in TS ligands binding. In addition, the Advanced Poisson-Boltzmann Solver (APBS) results show considerable differences in the distribution of electrostatic potential around Trp 103, as compared to distributions observed for all remaining Trp residues in the mTS family of structures. Together, spectroscopic and APBS results reveal a possible interplay between Trp 103 and His190, which contributes to a reduction in enzymatic activity in the case of H190A mutation. Comparison of electrostatic potential for mTS complexes, and their mutants, with the substrate, dUMP, and inhibitors, FdUMP and N4-OH-dCMP, suggests its weaker influence on the enzyme-ligand interactions in N4OH-dCMP-mTS compared to dUMP-mTS and FdUMP-mTS complexes. This difference may be crucial for the explanation of the "abortive reaction" inhibitory mechanism of N4OH-dCMP towards TS. In addition, based on structural analyses and the H190A mutant capacity to form a denaturation-resistant complex with N4-OH-dCMP in the mTHF-dependent reaction, His190 is apparently responsible for a strong preference of the enzyme active center for the anti rotamer of the imino inhibitor form.

Synthesis of Polyanionic C5-Modified 2'-Deoxyuridine and 2'-Deoxycytidine-5'-Triphosphates and Their Properties as Substrates for DNA Polymerases.

Modified 2'-deoxyribonucleotide triphosphates (dNTPs) have widespread applications in both existing and emerging biomolecular technologies. For such applications it is an essential requirement that the modified dNTPs be substrates for DNA polymerases. To date very few examples of C5-modified dNTPs bearing negatively charged functionality have been described, despite the fact that such nucleotides might potentially be valuable in diagnostic applications using Si-nanowire-based detection systems. Herein we have synthesised C5-modified dUTP and dCTP nucleotides each of which are labelled with an dianionic reporter group. The reporter group is tethered to the nucleobase via a polyethylene glycol (PEG)-based linkers of varying length. The substrate properties of these modified dNTPs with a variety of DNA polymerases have been investigated to study the effects of varying the length and mode of attachment of the PEG linker to the nucleobase. In general, nucleotides containing the PEG linker tethered to the nucleobase via an amide rather than an ether linkage proved to be the best substrates, whilst nucleotides containing PEG linkers from PEG6 to PEG24 could all be incorporated by one or more DNA polymerase. The polymerases most able to incorporate these modified nucleotides included Klentaq, Vent(exo-) and therminator, with incorporation by Klenow(exo-) generally being very poor.

Intrinsic Fluorescence of the Active and the Inactive Functional Forms of Human Thymidylate Synthase.

The observables associated with protein intrinsic fluorescence - spectra, time decays, anisotropies - offer opportunities to monitor in real time and non-invasively a protein's functional form and its interchange with other forms with different functions. We employed these observables to sketch the fluorometric profiles of two functional forms of human thymidylate synthase (hTS), a homodimeric enzyme crucial for cell proliferation and thus targeted by anticancer drugs. The protein takes an active and an inactive form. Stabilization of the latter by peptides that, unlike classical hTS inhibitors, bind it at the monomer/monomer interface offers an alternative inhibition mechanism that promises to avoid the onset of drug resistance in anticancer therapy. The fluorescence features depicted herein can be used as tools to identify and quantify each of the two protein forms in solution, thus making it possible to investigate the kinetic and thermodynamic aspects of the active/inactive conformational interchange. Two examples of fluorometrically monitored interconversion kinetics are provided.

Structure analysis of thymidylate synthase from white spot syndrome virus reveals WSSV-specific structural elements.

White spot syndrome virus (WSSV), the causative agent of white spot disease (WSD) severely affecting crustacean life forms, is highly contagious and forms the principal cause of massive economic losses in the shrimp aquaculture industry. Previous studies have demonstrated thymidylate synthase as a successful anti-cancer therapeutic drug target, leading to various anti-cancer drugs. The differential utilization of nucleotide precursors between white spot syndrome virus and shrimp encouraged us to analyze WSSV-thymidylate synthase (wTS). Here, we report the crystal structures of wTS in its apo-form and as a ternary complex with deoxyuridine monophosphate (dUMP) and methotrexate at a resolution of 2.35 Å and 2.6 Å, respectively. wTS possesses a fold characteristic to known thymidylate synthase (TS) structures. Like other TS structures, the apo-form of wTS displays an open conformation, whereas the wTS ternary complex attains a closed conformation. While the C-terminal loop maintains a typical distance from methotrexate, the Sγ atom of the catalytic Cys is positioned farther from the C6 atom of dUMP. Altogether, we report the first TS structure from a crustacean virus and highlight its distinction from shrimp and other TS structures.

Identification of novel regulators of dendrite arborization using cell type-specific RNA metabolic labeling.

Obtaining neuron transcriptomes is challenging; their complex morphology and interconnected microenvironments make it difficult to isolate neurons without potentially altering gene expression. Multidendritic sensory neurons (md neurons) of Drosophila larvae are commonly used to study peripheral nervous system biology, particularly dendrite arborization. We sought to test if EC-tagging, a biosynthetic RNA tagging and purification method that avoids the caveats of physical isolation, would enable discovery of novel regulators of md neuron dendrite arborization. Our aims were twofold: discover novel md neuron transcripts and test the sensitivity of EC-tagging. RNAs were biosynthetically tagged by expressing CD:UPRT (a nucleobase-converting fusion enzyme) in md neurons and feeding 5-ethynylcytosine (EC) to larvae. Only CD:UPRT-expressing cells are competent to convert EC into 5-ethynyluridine-monophosphate which is subsequently incorporated into nascent RNA transcripts. Tagged RNAs were purified and used for RNA-sequencing. Reference RNA was prepared in a similar manner using 5-ethynyluridine (EUd) to tag RNA in all cells and negative control RNA-seq was performed on "mock tagged" samples to identify non-specifically purified transcripts. Differential expression analysis identified md neuron enriched and depleted transcripts. Three candidate genes encoding RNA-binding proteins (RBPs) were tested for a role in md neuron dendrite arborization. Loss-of-function for the m6A-binding factor Ythdc1 did not cause any dendrite arborization defects while RNAi of the other two candidates, the poly(A) polymerase Hiiragi and the translation regulator Hephaestus, caused significant defects in dendrite arborization. This work provides an expanded view of transcription in md neurons and a technical framework for combining EC-tagging with RNA-seq to profile transcription in cells that may not be amenable to physical isolation.

Metabolic labeling of Escherichia coli genomic DNA with erythrosine-11-dUTP for functional imaging via correlative microscopy.

The fluorescent metabolic labeling of microorganisms genome is an advanced imaging technique to observe and study the native shapes, structural changes, functions, and tracking of nucleic acids in single cells or tissues. We have attempted to visualize the newly synthesized DNA within the intact nucleoid of ice-embedded proliferating cells of Escherichia coli K-12 (thymidine-requiring mutant, strain N4316) via correlative light-electron microscopy. For that purpose, erythrosine-11-dUTP was synthesized and used as a modified analog of the exogenous thymidine substrate for metabolic incorporation into the bacterial chromosome. The formed fluorescent genomic DNA during in cellulo polymerase reaction caused a minimal cellular arrest and cytotoxicity of E. coli at certain controlled conditions. The stained cells were visualized in typical red emission color via an epifluorescence microscope. They were further ice-embedded and examined with a Hilbert differential contrast transmission electron microscopy. At high-resolution, the ultrastructure of tagged nucleoid appeared with significantly higher electron dense in comparison to the unlabeled one. The enhanced contrast areas in the chromosome were ascribed to the presence of iodine contents from erythrosine dye. The presented labeling approach might be a powerful strategy to reveal the structural and dynamic changes in natural DNA replication including the relationship between newly synthesized in vivo nucleic acid and the physiological state of the cell.

PCR incorporation of dUMPs modified with aromatic hydrocarbon substituents of different hydrophilicities: Synthesis of C5-modified dUTPs and PCR studies using Taq, Tth, Vent (exo-) and Deep Vent (exo-) polymerases.

Deoxyuridine triphosphate derivatives (dUTPs) modified at the C5 position of the pyrimidine ring with various aromatic hydrocarbon substituents of different hydrophilicities have been synthesized. The aromatic hydrocarbon substituents were attached to dUTPs via a CHCHCH2NHCOCH2 linker. The efficiency of the PCR incorporation of modified dUMPs using Taq, Tth, Vent (exo-) and Deep Vent (exo-) polymerases and a model DNA template containing one, two and three adjacent adenine nucleotides at three different sites within the sequence was investigated. For all the polymerases used, the yield of the modified PCR product was significantly increased with increasing hydrophilicity of the aromatic hydrocarbon substituent. In particular, for the above polymerases, the efficiency of the incorporation of dUMPs modified with the most hydrophilic of the studied aromatic hydrocarbon substituents, a 4-hydroxyphenyl residue, was 60-85% of the efficiency of dTMP incorporation. At the same time, the relative efficiencies of the incorporation of dUMPs modified with 2-, 4-methoxyphenyl, phenyl and 4-nitrophenyl substituents ranged from 20 to 50% and were 2-18% for the 1-naphthalene and 4-biphenyl groups, which were the most hydrophobic of the studied aromatic hydrocarbon substituents.

Chemoenzymatic Preparation of Functional Click-Labeled Messenger RNA.

Synthetic mRNAs are promising candidates for a new class of transformative drugs that provide genetic information for patients' cells to develop their own cure. One key advancement to develop so-called druggable mRNAs was the preparation of chemically modified mRNAs, by replacing standard bases with modified bases, such as uridine with pseudouridine, which can ameliorate the immunogenic profile and translation efficiency of the mRNA. Thus the introduction of modified nucleobases was the foundation for the clinical use of such mRNAs. Herein we describe modular and simple methods to chemoenzymatically modify mRNA. Alkyne- and/or azide-modified nucleotides are enzymatically incorporated into mRNA and subsequently conjugated to fluorescent dyes using click chemistry. This allows visualization of the labeled mRNA inside cells. mRNA coding for the enhanced green fluorescent protein (eGFP) was chosen as a model system and the successful expression of eGFP demonstrated that our modified mRNA is accepted by the translation machinery.

Quantitation of deoxynucleoside triphosphates by click reactions.

The levels of the four deoxynucleoside triphosphates (dNTPs) are under strict control in the cell, as improper or imbalanced dNTP pools may lead to growth defects and oncogenesis. Upon treatment of cancer cells with therapeutic agents, changes in the canonical dNTPs levels may provide critical information for evaluating drug response and mode of action. The radioisotope-labeling enzymatic assay has been commonly used for quantitation of cellular dNTP levels. However, the disadvantage of this method is the handling of biohazard materials. Here, we described the use of click chemistry to replace radioisotope-labeling in template-dependent DNA polymerization for quantitation of the four canonical dNTPs. Specific oligomers were designed for dCTP, dTTP, dATP and dGTP measurement, and the incorporation of 5-ethynyl-dUTP or C8-alkyne-dCTP during the polymerization reaction allowed for fluorophore conjugation on immobilized oligonucleotides. The four reactions gave a linear correlation coefficient >0.99 in the range of the concentration of dNTPs present in 106 cells, with little interference of cellular rNTPs. We present evidence indicating that data generated by this methodology is comparable to radioisotope-labeling data. Furthermore, the design and utilization of a robust microplate assay based on this technology evidenced the modulation of dNTPs in response to different chemotherapeutic agents in cancer cells.

Fluorescence In Situ Hybridization and Rehybridization Using Bacterial Artificial Chromosome Probes.

Fluorescence in situ hybridization (FISH) method enables in situ genetic analysis of both metaphase and interphase cells from different types of material, including cell lines, cell smears, and fresh and paraffin-embedded tissue. Despite the growing number of commercially available FISH probes, still for large number of gene loci or chromosomal regions commercial probes are not available. Here we describe a simple method for generating FISH probes using bacterial artificial chromosomes (BAC). Due to genome-wide coverage of BAC clones, there are almost unlimited possibilities for the analysis of any genomic regions using BAC FISH probes.

Triazine-Modified 7-Deaza-2'-deoxyadenosines: Better Suited for Bioorthogonal Labeling of DNA by PCR than 2'-Deoxyuridines.

6-Ethynyl-1,2,4-triazine is a small bioorthogonally reactive group we applied for fluorescent labeling of oligonucleotides by Diels-Alder reactions with inverse electron demand. We synthetically attached this functional group to the 7-position of 7-deaza-2'-deoxyadenosine triphosphate and to the 5-position of 2'-deoxyuridine triphosphate. Both modified nucleotide triphosphates were used in comparison for primer extension experiments (PEX) and PCR amplification to finally yield multilabeled oligonucleotides by the postsynthetic reaction with a highly reactive bicyclo[6.1.0]nonyne-rhodamine conjugate. These experiments show that 6-ethynyl-1,2,4-triazine is much better tolerated by the DNA polymerase when attached to the 7-position of 7-deaza-2'-deoxyadenosine in comparison to the attachment at the 5-position of 2'-deoxyuridine. This became evident both by PAGE analysis of the PCR products and real-time kinetic observation of DNA polymerase activity during primer extension using switchSENSE. Generally, our results imply that bioorthogonal labeling strategies are better suited for 7-deaza-2'-adenosines than conventional and available 2'-deoxyuridines.

The etiology of uracil residues in the Saccharomyces cerevisiae genomic DNA.

Non-canonical residue in DNA is a major and conserved source of genome instability. The appearance of uracil residues in DNA accompanies a significant mutagenic consequence and is regulated at multiple levels, from the concentration of available dUTP in the nucleotide pool to the excision repair for removal from DNA. Recently, an interesting phenomenon of transcription-associated elevation in uracil-derived mutations was described in Saccharomyces cerevisiae genome. While trying to understand the variability in mutagenesis, we uncovered that the frequency of uracil incorporation into DNA can vary depending on the transcription rate and that the non-replicative, repair-associated DNA synthesis underlies the higher uracil density of the actively transcribed genomic loci. This novel mechanism brings together the chemical vulnerability of DNA under transcription and the uracil-associated mutagenesis, and has the potential to apply to other non-canonical residues of mutagenic importance.

Factors Affecting the Tailing of Blunt End DNA with Fluorescent Pyrimidine dNTPs.

The transferase activity of non-proofreading DNA polymerases is a well-known phenomenon that has been utilized in cloning and sequencing applications. The non-templated addition of modified nucleotides at DNA blunt ends is a potentially useful feature of DNA polymerases that can be used for selective transformation of DNA 3' ends. In this paper, we characterized the tailing reaction at perfectly matched and mismatched duplex ends with Cy3- and Cy5-modified pyrimidine nucleotides. It was shown that the best DNA tailing substrate does not have a perfect Watson-Crick base pair at the end. Mismatched duplexes with a 3' dC were the most efficient in the Taq DNA polymerase-catalysed tailing reaction with a Cy5-modified dUTP. We further demonstrated that the arrangement of the dye residue relative to the nucleobase notably affects the outcome of the tailing reaction. A comparative study of labelled deoxycytidine and deoxyuridine nucleotides showed higher efficiency for dUTP derivatives. The non-templated addition of modified nucleotides by Taq polymerase at a duplex blunt end was generally complicated by the pyrophosphorolysis and 5' exonuclease activity of the enzyme.

Parallel reaction pathways and noncovalent intermediates in thymidylate synthase revealed by experimental and computational tools.

Thymidylate synthase was one of the most studied enzymes due to its critical role in molecular pathogenesis of cancer. Nevertheless, many atomistic details of its chemical mechanism remain unknown or debated, thereby imposing limits on design of novel mechanism-based anticancer therapeutics. Here, we report unprecedented isolation and characterization of a previously proposed intact noncovalent bisubstrate intermediate formed in the reaction catalyzed by thymidylate synthase. Free-energy surfaces of the bisubstrate intermediates interconversions computed with quantum mechanics/molecular mechanics (QM/MM) methods and experimental assessment of the corresponding kinetics indicate that the species is the most abundant productive intermediate along the reaction coordinate, whereas accumulation of the covalent bisubstrate species largely occurs in a parallel nonproductive pathway. Our findings not only substantiate relevance of the previously proposed noncovalent intermediate but also support potential implications of the overstabilized covalent intermediate in drug design targeting DNA biosynthesis.

N-terminal domain of human uracil DNA glycosylase (hUNG2) promotes targeting to uracil sites adjacent to ssDNA-dsDNA junctions.

The N-terminal domain (NTD) of nuclear human uracil DNA glycosylase (hUNG2) assists in targeting hUNG2 to replication forks through specific interactions with replication protein A (RPA). Here, we explored hUNG2 activity in the presence and absence of RPA using substrates with ssDNA-dsDNA junctions that mimic structural features of the replication fork and transcriptional R-loops. We find that when RPA is tightly bound to the ssDNA overhang of junction DNA substrates, base excision by hUNG2 is strongly biased toward uracils located 21 bp or less from the ssDNA-dsDNA junction. In the absence of RPA, hUNG2 still showed an 8-fold excision bias for uracil located <10 bp from the junction, but only when the overhang had a 5' end. Biased targeting required the NTD and was not observed with the hUNG2 catalytic domain alone. Consistent with this requirement, the isolated NTD was found to bind weakly to ssDNA. These findings indicate that the NTD of hUNG2 targets the enzyme to ssDNA-dsDNA junctions using RPA-dependent and RPA-independent mechanisms. This structure-based specificity may promote efficient removal of uracils that arise from dUTP incorporation during DNA replication, or additionally, uracils that arise from DNA cytidine deamination at transcriptional R-loops during immunoglobulin class-switch recombination.

Synthesis of Dihydroxyalkynyl and Dihydroxyalkyl Nucleotides as Building Blocks or Precursors for Introduction of Diol or Aldehyde Groups to DNA for Bioconjugations.

(3,4-Dihydroxybut-1-ynyl)uracil, -cytosine and -7-deazaadenine 2'-deoxyribonucleoside triphosphates (dNTPs) were prepared by direct aqueous Sonogashira cross-coupling of halogenated dNTPs with dihydroxybut-1-yne and converted to 3,4-dihydroxybutyl dNTPs through catalytic hydrogenation. Sodium periodate oxidative cleavage of dihydroxybutyl-dUTP gave the desired aliphatic aldehyde-linked dUTP, whereas the oxidative cleavage of the corresponding deazaadenine dNTP gave a cyclic aminal. All dihydroxyalkyl or -alkynyl dNTPs and the formylethyl-dUTP were good substrates for DNA polymerases and were used for synthesis of diol- or aldehyde-linked DNA. The aldehyde linked DNA was used for the labelling or bioconjugations through hydrazone formation or reductive aminations.