Incorporation Of Analogs Research Articles

A T7 RNA Polymerase Mutant Enhances the Yield of 5′‐Thienoguanosine‐Initiated RNAs

Spectroscopic methods, which are used to establish RNA structure-function relationships, require strategies for post-synthetic, site-specific incorporation of chemical probes into target RNAs. For RNAs larger than 50 nt, the enzymatic incorporation of a nucleoside or nucleotide monophosphate guanosine analogue (G analogue) at their 5'-end is routinely achieved by T7 RNA polymerase (T7RNAP)-mediated in vitro transcription (IVT) of the appropriate DNA template containing a GTP-initiating class III Φ6.5 promoter. However, when high G analogue:GTP ratios are used to bias G analogue incorporation at the 5'-end, RNA yield is compromised. Here, we show that the use of a T7RNAP P266L mutant in IVT with 10:1 thienoguanosine (th G):GTP increased the percent incorporation and yield of 5'-th G-initiated precursor tRNA for a net ≈threefold gain compared to IVT with wild-type T7RNAP. We also demonstrated that a one-pot multienzyme approach, consisting of transcription by T7RNAP P266L and post-transcriptional cleanup by polyphosphatase and an exonuclease, led to essentially near-homogeneous 5'-th G-modified transcripts. This approach should be of broad utility in preparing 5'-modified RNAs.

Visualization of the native shape of bodipy-labeled DNA in Escherichia coli by correlative microscopy.

The native shape and intracellular distribution of newly synthesized DNA was visualized by correlative (light and electron) microscopy in ice embedded whole cells of Escherichia coli. For that purpose, the commercially available modified nucleoside triphosphate named BODIPY® FL-14-dUTP was enzymatically incorporated in vivo into the genome of E. coli mutant K12 strain, which cannot synthesize thymine. The successful incorporation of this thymidine analogue was confirmed first by fluorescence microscope, where the cells were stained in the typical for bodipy green color. Later the preselected labeled E. coli were observed by Hilbert Differential Transmission Electron Microscope (HDC TEM) and the distribution of elemental boron (contained in bodipy) was visualized at high-resolution by an electron spectroscopic imaging (ESI) technique. The practical detection limit of boron was found to be around 5 ∼ 10 mmol/kg in area of 0.1 μm2 , which demonstrated that ESI is a suitable approach to study the cytochemistry and location of labeled nucleic fragments within the cytoplasmic chromosomal area. In addition, the fine cellular fibrous and chromosomal ultrastructures were revealed in situ by combing of phase-plate HDC TEM and ESI. The obtained results conclude that the correlation between fluorescent microscopy with phase-plate HDC TEM and ESI is a powerful approach to explore the structural and conformation dynamics of DNA replication machinery in frozen cells close to the living state.

Spatial heterogeneity of glycogen and its metabolizing enzymes in Aspergillus nidulans hyphal tip cells

Glycogen is a homopolymer of glucose and a ubiquitous cellular-storage carbon. This study investigated which Aspergillus nidulans genes are involved in glycogen metabolism. Gene disruptants of predicted glycogen synthase (gsyA) and glycogenin (glgA) genes accumulated less cellular glycogen than the wild type strain, indicating that GsyA and GlgA synthesize glycogen similarly to other eukaryotes. Meanwhile, gene disruption of gphA encoding glycogen phosphorylase increased the amount of glycogen to a higher degree than wild type during the stationary phase that accompanies carbon-source limitation. GFP-tagged GsyA and GphA were distributed in the cytosol and formed punctate and filamentous structures, respectively. Carbon starvation resulted in elongated GphA-GFP filaments and increased numbers of filaments. These structures were more frequently located in the basal regions of tip cells and adjacent cells than in the apical regions of tip cells. Cellular glycogen visualized by incorporation of a fluorescent glucose analog accumulated in cytoplasmic puncta that were more prevalent in the basal regions, a pattern similar to that seen for GsyA. The colocalization of glycogen and GsyA at punctate structures in tip and sub-apical cells likely represents the cellular machinery for synthesizing glycogen. More frequent colocalization in the basal, rather than tip cell apical regions indicated that tip cells have differentiated subcellular regions for glycogen synthesis. Our findings regarding glycogen, GsyA and GphA distribution evoke the spatial heterogeneity of glycogen metabolism in fungal hyphae.

Contribution of cell proliferation to axial elongation in the red flour beetle Tribolium castaneum.

Most arthropods generate their posterior bodies by adding segments periodically, as the embryo grows, from a posteriorly located region called the segment addition zone. This mode of segmentation is shared with vertebrates and relies on oscillatory mechanisms, where the temporal periodicity of a clock is translated into repetitive spatial patterns. This ordered anterior-to-posterior pattern is achieved at the same time as the tissue elongates, opening the question of the functional coordination between the mechanisms of segmental patterning and posterior growth. The study of these processes in different arthropods has played an important role in unravelling some of the molecular mechanisms of segment formation. However, the behavior of cells during elongation and how cellular processes affect this segmental patterning has been poorly studied. Cell proliferation together with cell rearrangements are presumed to be the major forces driving axis elongation in the red flour beetle Tribolium castaneum. However, there still no strong evidence about the role and distribution of cell proliferation within the embryo. In this study, we propose to address these questions by using whole embryo cultures and pharmacological manipulation. We show that considerable cell proliferation occurs during germband elongation, measured by incorporation of the nucleoside analog of thymidine 5-Ethynyl-2’-deoxyuridine, EdU. Moreover, proliferating cells appeared to be spread along the elongating embryo with a posterior bias at early segmentation. In addition, when we blocked cell division, treated germbands were always shorter than controls and in some cases not able to fully elongate, even when control embryos already started to retract and leg buds are evident. Finally, we found that the absence of cell proliferation has no apparent effect on segmental patterning, as evidenced by Tc-engrailed (Tc-en) gene expression.

Signatures of Nucleotide Analog Incorporation by an RNA-Dependent RNA Polymerase Revealed Using High-Throughput Magnetic Tweezers

SummaryRNA viruses pose a threat to public health that is exacerbated by the dearth of antiviral therapeutics. The RNA-dependent RNA polymerase (RdRp) holds promise as a broad-spectrum, therapeutic target because of the conserved nature of the nucleotide-substrate-binding and catalytic sites. Conventional, quantitative, kinetic analysis of antiviral ribonucleotides monitors one or a few incorporation events. Here, we use a high-throughput magnetic tweezers platform to monitor the elongation dynamics of a prototypical RdRp over thousands of nucleotide-addition cycles in the absence and presence of a suite of nucleotide analog inhibitors. We observe multiple RdRp-RNA elongation complexes; only a subset of which are competent for analog utilization. Incorporation of a pyrazine-carboxamide nucleotide analog, T-1106, leads to RdRp backtracking. This analysis reveals a mechanism of action for this antiviral ribonucleotide that is corroborated by cellular studies. We propose that induced backtracking represents a distinct mechanistic class of antiviral ribonucleotides.

Click Chemistry for Analysis of Cell Proliferation in Flow Cytometry.

The measurement of cellular proliferation is fundamental to the assessment of cellular health, genotoxicity, and the evaluation of drug efficacy. Labeling, detection, and quantification of cells in the synthesis phase of cell cycle progression are not only important for characterizing basic biology, but also in defining cellular responses to drug treatments. Changes in DNA replication during S-phase can provide valuable insights into mechanisms of cell growth, cell cycle kinetics, and cytotoxicity. A common method for detection of cell proliferation is the incorporation of a thymidine analog during DNA synthesis. This chapter presents a pulse labeling method using the thymidine analog, 5-ethynyl-2'-deoxyuridine (EdU), with subsequent detection by click chemistry. EdU detection using click chemistry is bio-orthogonal to most living systems and does not non-specifically label other biomolecules. Live cells are first pulsed with EdU. After antibody labeling cell surface markers, fixation, and permeabilization, the incorporated EdU is covalently labeled using click chemistry thereby identifying proliferating cells. Improvements in click chemistry allow for labeling in the presence of fluorescent proteins and phycobiliproteins without quenching due to copper. Measuring DNA replication during cell cycle progression has cell health applications in flow cytometry, fluorescence microscopy, and high content imaging. This protocol has been developed and optimized for research use only and is not suitable for use in diagnostic procedures. © 2017 by John Wiley & Sons, Inc.

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions.

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods. Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

Sequential one-pot multienzyme synthesis of hyaluronan and its derivative

Hyaluronan (HA) is a linear polysaccharide composed of repeating disaccharide units. It has been well documented to play an array of biological functions in cancer events. Here, we reported a sequential one-pot multienzyme (OPME) strategy for in vitro synthesis of HA and its derivatives. The strategy, which combined in situ sugar nucleotides generation with HA chain polymerization, could convert cheap monosaccharides into HA polymers without consuming exogenous sugar nucleotide donors. HA polymers (number-average molecular weight ranged from 1.5×104 to 5.5×105Da) with over 70% yields were efficiently synthesized and purified from this one-pot system. More importantly, partial labeled HA derivative was further synthesized by metabolic incorporation of unnatural monosaccharide analogues into the sequential OPME system. Cross-linked HA hydrogel was achieved via copper (I)-catalyzed azide-alkyne cycloaddition and exhibited novel networks consisting of both inter- and intra-connected HA chains, which could facilitate the potential applications of this unique polysaccharide.

Intracellular Imaging of Protein-Specific Glycosylation.

Posttranslational protein glycosylation is conserved in all kingdoms of life and implicated in the regulation of protein structure, function, and localization. The visualization of glycosylation states of designated proteins within living cells is of great importance for unraveling the biological roles of intracellular protein glycosylation. Our generally applicable approach is based on the incorporation of a glucosamine analog, Ac4GlcNCyoc, into the cellular glycome via metabolic engineering. Ac4GlcNCyoc can be labeled in a second step via inverse-electron-demand Diels-Alder chemistry with fluorophores inside living cells. Additionally, target proteins can be expressed as enhanced green fluorescent protein (EGFP)-fusion proteins. To assess the proximity of the donor EGFP and the glycan-anchored acceptor fluorophore, Förster resonance energy transfer (FRET) is employed and read out with high contrast by fluorescence lifetime imaging (FLIM) microscopy. In this chapter, we present a detailed description of methods required to perform protein-specific imaging of glycosylation inside living cells. These include the complete synthesis of Ac4GlcNCyoc, immunoprecipitation of EGFP-fusion proteins to examine the Ac4GlcNCyoc modification state, and a complete section on basics, performance, as well as data analysis for FLIM-FRET microscopy. We also provide useful notes necessary for reproducibility and point out strengths and limitations of the approach.

Targeting Bacillus anthracis toxicity with a genetically selected inhibitor of the PA/CMG2 protein-protein interaction

The protein-protein interaction between the human CMG2 receptor and the Bacillus anthracis protective antigen (PA) is essential for the transport of anthrax lethal and edema toxins into human cells. We used a genetically encoded high throughput screening platform to screen a SICLOPPS library of 3.2 million cyclic hexapeptides for inhibitors of this protein-protein interaction. Unusually, the top 3 hits all contained stop codons in the randomized region of the library, resulting in linear rather than cyclic peptides. These peptides disrupted the targeted interaction in vitro; two act by binding to CMG2 while one binds PA. The efficacy of the most potent CMG2-binding inhibitor was improved through the incorporation of non-natural phenylalanine analogues. Cell based assays demonstrated that the optimized inhibitor protects macrophages from the toxicity of lethal factor.

A Glutamic Acid Analog Bearing an Ethylenediamine Moiety Promotes the Cytosolic Delivery of TAT Peptides

The stepwise protonation of ethylenediamine-containing peptides at low pH in endosomes is hypothesized to disrupt the endosomal membrane and enhance cytosolic delivery. Incorporation of a novel glutamic acid analog bearing an ethylenediamine moiety into TAT peptides markedly enhanced cytosolic delivery without cytotoxicity.

Imaging HIV-1 Genomic DNA from Entry through Productive Infection.

In order to track the fate of HIV-1 particles from early entry events through productive infection, we developed a method to visualize HIV-1 DNA reverse transcription complexes by the incorporation and fluorescent labeling of the thymidine analog 5-ethynyl-2'-deoxyuridine (EdU) into nascent viral DNA during cellular entry. Monocyte-derived macrophages were chosen as natural targets of HIV-1, as they do not divide and therefore do not incorporate EdU into chromosomal DNA, which would obscure the detection of intranuclear HIV-1 genomes. Using this approach, we observed distinct EdU puncta in the cytoplasm of infected cells within 12 h postinfection and subsequent accumulation of puncta in the nucleus, which remained stable through 5 days. The depletion of the restriction factor SAMHD1 resulted in a markedly increased number of EdU puncta, allowing efficient quantification of HIV-1 reverse transcription events. Analysis of HIV-1 isolates bearing defined mutations in the capsid protein revealed differences in their cytoplasmic and nuclear accumulation, and data from quantitative PCR analysis closely recapitulated the EdU results. RNA fluorescence in situ hybridization identified actively transcribing, EdU-labeled HIV-1 genomes in productively infected cells, and immunofluorescence analysis confirmed that CDK9, phosphorylated at serine 175, was recruited to RNA-positive HIV-1 DNA, providing a means to directly observe transcriptionally active HIV-1 genomes in productively infected cells. Overall, this system allows stable labeling and monitoring of HIV genomic DNA within infected cells during cytoplasmic transit, nuclear import, and mRNA synthesis.IMPORTANCE The fates of HIV-1 reverse transcription products within infected cells are not well understood. Although previous imaging approaches identified HIV-1 intermediates during early stages of infection, few have connected these events with the later stages that ultimately lead to proviral transcription and the production of progeny virus. Here we developed a technique to label HIV-1 genomes using modified nucleosides, allowing subsequent imaging of cytoplasmic and nuclear HIV-1 DNA in infected monocyte-derived macrophages. We used this technique to track the efficiency of nuclear entry as well as the fates of HIV-1 genomes in productively and nonproductively infected macrophages. We visualized transcriptionally active HIV-1 DNA, revealing that transcription occurs in a subset of HIV-1 genomes in productively infected cells. Collectively, this approach provides new insights into the nature of transcribing HIV-1 genomes and allows us to track the entire course of infection in macrophages, a key target of HIV-1 in infected individuals.

Translesion Synthesis DNA Polymerase Kappa Is Indispensable for DNA Repair Synthesis in Cisplatin Exposed Dorsal Root Ganglion Neurons.

In the peripheral nervous system (PNS) in the absence of tight blood barrier, neurons are at increased risk of DNA damage, yet the question of how effectively PNS neurons manage DNA damage remains largely unanswered. Genotoxins in systemic circulation include chemotherapeutic drugs that reach peripheral neurons and damage their DNA. Because neurotoxicity of platinum-based class of chemotherapeutic drugs has been implicated in PNS neuropathies, we utilized an in vitro model of Dorsal Root Ganglia (DRGs) to investigate how peripheral neurons respond to cisplatin that forms intra- and interstrand crosslinks with their DNA. Our data revealed strong transcriptional upregulation of the translesion synthesis DNA polymerase kappa (Pol κ), while expression of other DNA polymerases remained unchanged. DNA Pol κ is involved in bypass synthesis of diverse DNA lesions and considered a vital player in cellular survival under injurious conditions. To assess the impact of Pol κ deficiency on cisplatin-exposed DRG neurons, Pol κ levels were reduced using siRNA. Pol κ targeting siRNA diminished the cisplatin-induced nuclear Pol κ immunoreactivity in DRG neurons and decreased the extent of cisplatin-induced DNA repair synthesis, as reflected in reduced incorporation of thymidine analog into nuclear DNA. Moreover, Pol κ depletion exacerbated global transcriptional suppression induced by cisplatin in DRG neurons. Collectively, these findings provide the first evidence for critical role of Pol κ in DNA damage response in the nervous system and call attention to implications of polymorphisms that modify Pol κ activity, on maintenance of genomic integrity and neuronal function in exogenously challenged PNS.

Modelling T cell proliferation: Dynamics heterogeneity depending on cell differentiation, age, and genetic background.

Cell proliferation is the common characteristic of all biological systems. The immune system insures the maintenance of body integrity on the basis of a continuous production of diversified T lymphocytes in the thymus. This involves processes of proliferation, differentiation, selection, death and migration of lymphocytes to peripheral tissues, where proliferation also occurs upon antigen recognition. Quantification of cell proliferation dynamics requires specific experimental methods and mathematical modelling. Here, we assess the impact of genetics and aging on the immune system by investigating the dynamics of proliferation of T lymphocytes across their differentiation through thymus and spleen in mice. Our investigation is based on single-cell multicolour flow cytometry analysis revealing the active incorporation of a thymidine analogue during S phase after pulse-chase-pulse experiments in vivo, versus cell DNA content. A generic mathematical model of state transition simulates through Ordinary Differential Equations (ODEs) the evolution of single cell behaviour during various durations of labelling. It allows us to fit our data, to deduce proliferation rates and estimate cell cycle durations in sub-populations. Our model is simple and flexible and is validated with other durations of pulse/chase experiments. Our results reveal that T cell proliferation is highly heterogeneous but with a specific “signature” that depends upon genetic origins, is specific to cell differentiation stages in thymus and spleen and is altered with age. In conclusion, our model allows us to infer proliferation rates and cell cycle phase durations from complex experimental 5-ethynyl-2'-deoxyuridine (EdU) data, revealing T cell proliferation heterogeneity and specific signatures.

Re-mineralizing dentin using an experimental tricalcium silicate cement with biomimetic analogs

ObjectivesTo characterize the re-mineralization potential of an experimental zirconium oxide (ZrO2) containing tricalcium silicate (TCS) cement, TCS 50, with the incorporation of biomimetic analogs at demineralized dentin. MethodsClass-I cavities were prepared in non- carious human third molars. The dentin cavities were demineralized using a pH-cycling protocol, involving 50 cyclic immersions in pH-4.8 and pH-7 baths for 0.5h and 2.5h, successively. The cavities were filled with TCS 50 with/without biomimetic analogs (3% polyacrylic acid, 8% sodium trimetaphosphate) being added to the mixed TCS 50 cement prior to application. The commercial hCSCs Biodentine (Septodont) and ProRoot MTA (Dentsply Sirona) served as controls. After 1 and 6 weeks storage in simulated body fluid (SBF), the polished specimen cross-sections were chemically characterized using a field-emission-gun Electron Probe Micro-Analysis (Feg- EPMA). ResultsEPMA line-scans and elemental mappings confirmed early re-mineralization induced by TCS 50 at 1 week. When biomimetic analogs were added to TCS 50, re-mineralization was more efficient after 6 weeks; the relative depth and intensity of re-mineralization were 79.7% and 76.6%, respectively, being significantly greater than at 1 week (pSignificance: The experimental TCS-based cement, TCS 50, proved to be capable of re-mineralizing artificially demineralized dentin. The incorporation of biomimetic analogs promoted re- mineralization upon 6-week SBF storage. However, re-mineralization appeared incomplete, this even for TCS 50 to which biomimetic analogs were added and upon 6-week SBF storage.

Analysis of Ribonucleotide 5'-Triphosphate Analogs as Potential Inhibitors of Zika Virus RNA-Dependent RNA Polymerase by Using Nonradioactive Polymerase Assays.

Zika virus (ZIKV) is an emerging human pathogen that is spreading rapidly through the Americas and has been linked to the development of microcephaly and to a dramatically increased number of Guillain-Barré syndrome cases. Currently, no vaccine or therapeutic options for the prevention or treatment of ZIKV infections exist. In the study described in this report, we expressed, purified, and characterized full-length nonstructural protein 5 (NS5) and the NS5 polymerase domain (NS5pol) of ZIKV RNA-dependent RNA polymerase. Using purified NS5, we developed an in vitro nonradioactive primer extension assay employing a fluorescently labeled primer-template pair. Both purified NS5 and NS5pol can carry out in vitro RNA-dependent RNA synthesis in this assay. Our results show that Mn2+ is required for enzymatic activity, while Mg2+ is not. We found that ZIKV NS5 can utilize single-stranded DNA but not double-stranded DNA as a template or a primer to synthesize RNA. The assay was used to compare the efficiency of incorporation of analog 5'-triphosphates by the ZIKV polymerase and to calculate their discrimination versus that of natural ribonucleotide triphosphates (rNTPs). The 50% inhibitory concentrations for analog rNTPs were determined in an alternative nonradioactive coupled-enzyme assay. We determined that, in general, 2'-C-methyl- and 2'-C-ethynyl-substituted analog 5'-triphosphates were efficiently incorporated by the ZIKV polymerase and were also efficient chain terminators. Derivatives of these molecules may serve as potential antiviral compounds to be developed to combat ZIKV infection. This report provides the first characterization of ZIKV polymerase and demonstrates the utility of in vitro polymerase assays in the identification of potential ZIKV inhibitors.

Synthesis of antisense oligonucleotides containing acyclic alkynyl nucleoside analogs and their biophysical and biological properties

The synthesis of oligonucleotide (ON) analogs, which can be used as antisense molecules, has recently gained much attention. Here, we report the synthesis and properties of an ON analog containing acyclic thymidine and cytidine analogs with a 4-pentyl-1,2-diol instead of the d-ribofuranose moiety. The incorporation of these analogs into the ON improved its nuclease resistance to 3′-exonucleases. Furthermore, it was found that the incorporation of the acyclic thymidine analog into a DNA/RNA duplex accelerates the RNA cleavage of a DNA/RNA duplex by Escherichia coli RNase H.

Synthesis, Photophysical Properties, and Enzymatic Incorporation of an Emissive Thymidine Analogue

Herein, we describe the synthesis, photophysical characterization, and DNA incorporation of a fluorescent nucleobase analogue, thieno[3,4-d]-pyrimidine T-mimic deoxynucleoside, thdT. thdT exhibits comparable thermal stability and selectivity of base pairing to that of natural T and noteworthy photophysical behaviors depending on its microenvironments in DNA. A thio-analogue of thymidine triphosphate, thdTTP, has been synthesized and enzymatically incorporated into DNA using naturally occurring polymerases and replication systems with assistance from dTTP. The present results show the utility of thdT as a fluorescent surrogate of thymidine for further applications.

Molecular bases for phyllomorph development in a one-leaf plant, Monophyllaea glabra.

The aboveground part of one-leaf plants (Gesneriaceae) consists of a unique shoot-like unit called a phyllomorph, which is composed of a growing lamina and one petiolode (a stem- and petiole-like organ). The phyllomorph has three meristems: a basal meristem (BM; involved in lamina growth), a petiolode meristem (PM; required for midrib and petiolode growth), and a groove meristem (GM; contributes to inflorescence and new phyllomorph development). Although the GM has a tunica-corpus structure like a conventional shoot apical meristem (SAM), the vegetative GM of a one-leaf plant does not form any organ primordia and has been considered a defective or suppressed SAM. In this study, we evaluated the nature of the three meristems to reconsider their roles in phyllomorph development in the one-leaf species Monophyllaea glabra. Mitotic activities of meristem cells were monitored by incorporation of a thymidine analog (EdU) into DNA. Orthologs of SHOOTMERISTEMLESS (STM) and PHANTASTICA, ROUGH SHEATH2, ASYMMETRIC LEAVES1 (PHAN/RS2/AS1) were isolated from M. glabra, and their expression patterns were investigated by quantitative reverse transcription polymerase chain reaction in combination with a laser microdissection technique. Mitotic activities were detected in all three phyllomorph meristems. Strong expression of the STM and PHAN/RS2/AS1 orthologs was detected in the vegetative GM and BM, respectively. The vegetative GM is an active meristem that expresses the STM ortholog. We postulate that the GM is required for growth of a phyllomorph by providing undifferentiated cells and/or growth regulators to the BM and/or PM.

Amplification of unscheduled DNA synthesis signal enables fluorescence-based single cell quantification of transcription-coupled nucleotide excision repair.

Nucleotide excision repair (NER) comprises two damage recognition pathways: global genome NER (GG-NER) and transcription-coupled NER (TC-NER), which remove a wide variety of helix-distorting lesions including UV-induced damage. During NER, a short stretch of single-stranded DNA containing damage is excised and the resulting gap is filled by DNA synthesis in a process called unscheduled DNA synthesis (UDS). UDS is measured by quantifying the incorporation of nucleotide analogues into repair patches to provide a measure of NER activity. However, this assay is unable to quantitatively determine TC-NER activity due to the low contribution of TC-NER to the overall NER activity. Therefore, we developed a user-friendly, fluorescence-based single-cell assay to measure TC-NER activity. We combined the UDS assay with tyramide-based signal amplification to greatly increase the UDS signal, thereby allowing UDS to be quantified at low UV doses, as well as DNA-repair synthesis of other excision-based repair mechanisms such as base excision repair and mismatch repair. Importantly, we demonstrated that the amplified UDS is sufficiently sensitive to quantify TC-NER-derived repair synthesis in GG-NER-deficient cells. This assay is important as a diagnostic tool for NER-related disorders and as a research tool for obtaining new insights into the mechanism and regulation of excision repair.