An Excimer Clamp for Measuring Damaged Base Excision by the DNA Repair Enzyme NTH1.
Angewandte Chemie (International ed. in English)
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.
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)
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