All Publications


  • Efficient DNA fluorescence labeling via base excision trapping. Nature communications Jun, Y. W., Harcourt, E. M., Xiao, L., Wilson, D. L., Kool, E. T. 2022; 13 (1): 5043

    Abstract

    Fluorescence labeling of DNAs is broadly useful, but methods for labeling are expensive and labor-intensive. Here we describe a general method for fluorescence labeling of oligonucleotides readily and cost-efficiently via base excision trapping (BETr), employing deaminated DNA bases to mark label positions, which are excised by base excision repair enzymes generating AP sites. Specially designed aminooxy-substituted rotor dyes trap the AP sites, yielding high emission intensities. BETr is orthogonal to DNA synthesis by polymerases, enabling multi-uracil incorporation into an amplicon and in situ BETr labeling without washing. BETr also enables labeling of dsDNA such as genomic DNA at a high labeling density in a single tube by use of nick translation. Use of two different deaminated bases facilitates two-color site-specific labeling. Use of a multi-labeled DNA construct as a bright fluorescence tag is demonstrated through the conjugation to an antibody for imaging proteins. Finally, double-strand selectivity of a repair enzyme is harnessed in sensitive reporting on the presence of a target DNA or RNA in a mixture with isothermal turnover and single nucleotide specificity. Overall, the results document a convenient and versatile method for general fluorescence labeling of DNAs.

    View details for DOI 10.1038/s41467-022-32494-8

    View details for PubMedID 36028479

  • Enhancing Repair of Oxidative DNA Damage with Small-Molecule Activators of MTH1. ACS chemical biology Lee, Y., Onishi, Y., McPherson, L., Kietrys, A. M., Hebenbrock, M., Jun, Y. W., Das, I., Adimoolam, S., Ji, D., Mohsen, M. G., Ford, J. M., Kool, E. T. 2022

    Abstract

    Impaired DNA repair activity has been shown to greatly increase rates of cancer clinically. It has been hypothesized that upregulating repair activity in susceptible individuals may be a useful strategy for inhibiting tumorigenesis. Here, we report that selected tyrosine kinase (TK) inhibitors including nilotinib, employed clinically in the treatment of chronic myeloid leukemia, are activators of the repair enzyme Human MutT Homolog 1 (MTH1). MTH1 cleanses the oxidatively damaged cellular nucleotide pool by hydrolyzing the oxidized nucleotide 8-oxo-2'-deoxyguanosine (8-oxo-dG)TP, which is a highly mutagenic lesion when incorporated into DNA. Structural optimization of analogues of TK inhibitors resulted in compounds such as SU0448, which induces 1000 ± 100% activation of MTH1 at 10 muM and 410 ± 60% at 5 muM. The compounds are found to increase the activity of the endogenous enzyme, and at least one (SU0448) decreases levels of 8-oxo-dG in cellular DNA. The results suggest the possibility of using MTH1 activators to decrease the frequency of mutagenic nucleotides entering DNA, which may be a promising strategy to suppress tumorigenesis in individuals with elevated cancer risks.

    View details for DOI 10.1021/acschembio.2c00038

    View details for PubMedID 35830623

  • Fluorescent detection of RNA using a base excision reporter. FASEB journal : official publication of the Federation of American Societies for Experimental Biology Harcourt, E. M., Jun, Y. W., Wilson, D. L., Ledgerwood, E. D., Kool, E. T. 2022; 36 Suppl 1

    Abstract

    A fluorescent probe originally designed for the detection of abasic sites and monitoring of DNA repair has been successfully used in the detection of RNA. The strategy employs deoxyuridine in a DNA probe complementary to the target RNA; the RNA/DNA hybrid was found to be a substrate for uracil-DNA glycosylase. After excision of uracil, rapid oxime formation between the hydroxylamine probe and the abasic site leads to a ~10-fold increase in fluorescence in the presence of the target RNA. Additional signal can be gained by incorporating multiple deoxyuridine nucleotides through reverse transcription. Reverse transcription, base excision, and probe incorporation can be carried out concurrently in a single vessel.

    View details for DOI 10.1096/fasebj.2022.36.S1.L7412

    View details for PubMedID 35553594

  • Discrimination of Invasive Human Skin Tumor Using an Ultrafast ATP-Proton AND-Gate Probe. ACS sensors Jun, Y. W., Kim, K. H., Yang, Y. J., Jung, Y., Kim, K., Kim, H., Oh, B. H., Ahn, K. H. 2022

    Abstract

    Cancer cells undergo unscheduled proliferation resulting from dysregulation of the cell cycle, and hence, evaluation in tumor is of keen interest to examine the invasiveness and recurrence of cancer in the lesion. Molecular probes capable of discriminating actively growing tumor from resting ones remain unexplored despite their vast importance. Here, we describe a novel strategy to visualize invasive areas in tumor with a fluorescence probe that implements synergistic fluorescence response toward the slightly acidic environment of tumor and an ATP-abundant nature of actively growing cells. The probe has been designed for ultrafast detection of ATP with high specificity. We demonstrate its utility in visualizing invasive areas in tumor by distinguishing basal cell carcinomas and squamous cell carcinomas at their early stages by two-photon microscopy.

    View details for DOI 10.1021/acssensors.1c02712

    View details for PubMedID 35353484

  • Fluorescence Imaging of Mitochondrial DNA Base Excision Repair Reveals Dynamics of Oxidative Stress Responses. Angewandte Chemie (International ed. in English) Jun, Y. W., Albarran, E., Wilson, D. L., Ding, J., Kool, E. T. 2021

    Abstract

    Mitochondrial function in cells declines with aging and with neurodegeneration, due in large part to accumulated mutations in mitochondrial DNA (mtDNA) that arise from deficient DNA repair. However, measuring this repair activity is challenging. Here we employ a molecular approach for visualizing mitochondrial base excision repair (BER) activity in situ by use of a fluorescent probe ( UBER ) that reacts rapidly with AP sites resulting from BER activity. Administering the probe to cultured cells revealed signals that were localized to mitochondria, enabling selective observation of mtDNA BER intermediates. The probe showed elevated DNA repair activity under oxidative stress, and responded to suppression of glycosylase activity. Furthermore, the probe illuminated the time lag between the initiation of oxidative stress and the initial step of BER. Absence of MTH1 in cells resulted in elevated demand for BER activity upon extended oxidative stress, while the absence of OGG1 activity limited glycosylation capacity.

    View details for DOI 10.1002/anie.202111829

    View details for PubMedID 34851014

  • Control of RNA with quinone methide reversible acylating reagents. Organic & biomolecular chemistry Park, H. S., Jash, B., Xiao, L., Jun, Y. W., Kool, E. T. 2021

    Abstract

    Caging RNA by polyacylation (cloaking) has been developed recently as a simple and rapid method to control the function of RNAs. Previous approaches for chemical reversal of acylation (uncloaking) made use of azide reduction followed by amine cyclization, requiring 2-4 h for the completion of cyclization. In new studies aimed at improving reversal rates and yields, we have designed novel acylating reagents that utilize quinone methide (QM) elimination for reversal. The QM de-acylation reactions were tested with two bioorthogonally cleavable motifs, azide and vinyl ether, and their acylation and reversal efficiencies were assessed with NMR and mass spectrometry on model small-molecule substrates as well as on RNAs. Successful reversal both with phosphines and strained alkenes was documented. Among the compounds tested, the azido-QM compound A-3 displayed excellent de-acylation efficiency, with t1/2 for de-acylation of less than an hour using a phosphine trigger. To test its function in RNA caging, A-3 was successfully applied to control EGFP mRNA translation in vitro and in HeLa cells. We expect that this molecular caging strategy can serve as a valuable tool for biological investigation and control of RNAs both in vitro and in cells.

    View details for DOI 10.1039/d1ob01713f

    View details for PubMedID 34528657

  • A Study on Hypoxia Susceptibility of Organ Tissues by Fluorescence Imaging with a Ratiometric Nitroreductase Probe ACS SENSORS Sarkar, S., Lee, H., Ryu, H., Singha, S., Lee, Y., Reo, Y., Jun, Y., Kim, K., Kim, W., Ahn, K. 2021; 6 (1): 148-155

    Abstract

    Hypoxia, a condition of oxygen deficiency in tissues, features various diseases including solid tumor. Under hypoxia, several reductases such as nitroreductases are elevated. Based on this fact, we have investigated an indirect way to assess the hypoxia susceptibility of different organ tissues (mouse lung, heart, spleen, kidney, and liver) by detecting nitroreductase present within. Among the organs, the kidney showed a notable susceptibility to hypoxia, which was due to the renal medulla, not due to the renal cortex, as observed by ratiometric fluorescence imaging with a probe. The probe features ratiometric signaling, NIR-emitting, two-photon absorbing, and pH-insensitive emission properties, offering a practical tool for studying the nitroreductase activity and, furthermore, hypoxia-associated biological processes.

    View details for DOI 10.1021/acssensors.0c01989

    View details for Web of Science ID 000613247400017

    View details for PubMedID 33334101

  • Environment-insensitive two-photon ratiometric probe for in cellulo quantitative measurement of hydrogen peroxide RESULTS IN CHEMISTRY Cho, S., Jun, Y., Reo, Y., Sarkar, S., Ahn, K. 2021; 3
  • DNA tiling enables precise acylation-based labeling and control of mRNA. Angewandte Chemie (International ed. in English) Xiao, L., Jun, Y. W., Kool, E. T. 2021

    Abstract

    Methods for site-selective labeling of long, native RNAs are needed for studying mRNA biology and future therapies. Current approaches involve engineering RNA sequences, which may alter folding, or are limited to specific sequences or bases. Here, we describe a versatile strategy for mRNA conjugation via a novel DNA tiling approach. The method, TRAIL, exploits a pool of "protector" oligodeoxynucleotides to hybridize and block the mRNA, combined with an "inducer" DNA that extrudes a reactive RNA loop for acylation at a predetermined site. Using TRAIL, an azido-acylimidazole reagent was employed for labeling and controlling RNA for multiple applications in vitro and in cells, including analysis of RNA-binding proteins, imaging mRNA in cells, and analysis and control of translation. The TRAIL approach offers an efficient and accessible way to label and manipulate RNAs of virtually any length or origin without altering native sequence.

    View details for DOI 10.1002/anie.202112106

    View details for PubMedID 34624169

  • A systematic study on the discrepancy of fluorescence properties between in solutions and in cells: super-bright, environment-insensitive benzocoumarin dyes. Chemical communications (Cambridge, England) Reo, Y. J., Jun, Y. W., Cho, S. W., Jeon, J., Roh, H., Singha, S., Dai, M., Sarkar, S., Kim, H. R., Kim, S., Jin, Y., Jung, Y. L., Yang, Y. J., Ban, C., Joo, J., Ahn, K. H. 2020

    Abstract

    The benzocoumarin dyes fluoresce negligibly in aqueous media but very strongly in cells, whereas representative conventional dyes display contrasting behaviour; the distinct emission behaviour of the fluorophores in organic solutions, in aqueous media, and in cell convinces the uniqueness of the cellular environment. The in cellulo superbright benzocoumarins also reveal an environment-insensitive emission behaviour, which is required for the reliable analysis via ratiometric imaging.

    View details for DOI 10.1039/d0cc03586f

    View details for PubMedID 32785337

  • Small Substrate or Large? Debate Over the Mechanism of Glycation Adduct Repair by DJ-1. Cell chemical biology Jun, Y. W., Kool, E. T. 2020

    Abstract

    Glycation, the term for non-enzymatic covalent reactions between aldehyde metabolites and nucleophiles on biopolymers, results in deleterious cellular damage and diseases. Since Parkinsonism-associated protein DJ-1 was proposed as a novel deglycase that directly repairs glycated adducts, it has been considered a major contributor to glycation damage repair. Recently, an interesting debate over the mechanism of glycation repair by DJ-1 has emerged, focusing on whether the substrate of DJ-1 is glycated adducts or the free small aldehydes. The physiological significance of DJ-1 on glycation defense also remains in question. This debate is complicated by the fact that glycated biomolecular adducts are in rapid equilibrium with free aldehydes. Here, we summarize experimental evidence for the two possibilities, highlighting both consistencies and conflicts. We discuss the experimental complexities from a mechanistic perspective, and suggest classes of experiments that should help clarify this debate.

    View details for DOI 10.1016/j.chembiol.2020.07.016

    View details for PubMedID 32783963

  • An Excimer Clamp for Measuring Damaged Base Excision by the DNA Repair Enzyme NTH1. Angewandte Chemie (International ed. in English) Jun, Y. W., Wilson, D. L., Kietrys, A. M., Lotsof, E. R., Conlon, S. G., David, S. S., Kool, E. T. 2020

    Abstract

    Direct measurement of DNA repair enzyme activities is important both for basic study of cellular repair pathways as well as for potential new translational applications in their associated diseases. NTH1, a major glycosylase targeting oxidized pyrimidines, prevents mutations arising from this damage, and the regulation of NTH1 activity is important in resisting oxidative stress and in suppressing tumor formation. Here we describe a novel molecular strategy for the direct detection of damaged DNA base excision activity by a ratiometric fluorescence change.This strategy utilizes glycosylation-induced excimer formation of pyrenes, and modified DNA probes incorporating two pyrene deoxynucleotides and a damaged base enable the direct, real-time detection of NTH1 activity in vitro and in cellular lysates. The probe design was also applied in screening for potential NTH1 inhibitors, leading to identification of a new small-molecule inhibitor with sub-micromolar potency.

    View details for DOI 10.1002/anie.202001516

    View details for PubMedID 32109332

  • Ratiometric Imaging of γ-Glutamyl Transpeptidase Unperturbed by pH, Polarity, and Viscosity Changes: A Benzocoumarin-Based Two-Photon Fluorescent Probe. Analytical chemistry Reo, Y. J., Jun, Y. W., Sarkar, S. n., Dai, M. n., Ahn, K. H. 2019

    Abstract

    γ-Glutamyltransferase (GGT) is involved in maintaining the intracellular glutathione levels and, at its elevated levels, is associated with various diseases including cancer and myocardial infarction. To study this enzyme in biological systems, fluorescent probes have received significant attention recently. As fluorescence signal is sensitive to environmental fluctuations; however, it is challenging to address the signal fluctuation issue. Disclosed is the benzocoumarin-based probe that enables ratiometric imaging of GGT activity levels in cells as well as in tissues, essentially unperturbed by medium pH, viscosity, and polarity changes. Validity of the probe is demonstrated by determining the GGT activity level in HeLa cells directly through ratiometric imaging. Furthermore, the probe and its enzymatic product are two-photon absorbing, extending its applicability to tissue: an 8.5-fold higher level of GGT in cancerous tissue over the normal tissue is determined, and the GGT activity levels between different mouse organ tissues are quantitatively compared with the highest level in the kidney. The probe with practicality holds great promise for studying GGT-associated biological processes directly through ratiometric imaging by two-photon microscopy.

    View details for DOI 10.1021/acs.analchem.9b03942

    View details for PubMedID 31566966

  • A fluorescent hydrazone exchange probe of pyridoxal phosphate for the assessment of vitamin B6 status. Chemical communications (Cambridge, England) Jun, Y. W., Hebenbrock, M. n., Kool, E. T. 2019

    Abstract

    Abnormal vitamin B6 status, marked by deficient intracellular concentrations of pyridoxal phosphate (PLP), is classified as a direct biomarker based on its biomedical significance. However, there exist no direct methods for measuring vitamin B6 status in intact cells. Here we describe the development of a fluorogenic probe, RAB6, which shows remarkable selectivity for PLP among the B6 vitamers and other cellular aldehydes.

    View details for DOI 10.1039/c9cc08458d

    View details for PubMedID 31808778