Genomic DNA i-motifs as fast sensors responsive to near-physiological pH microchanges.
Biosens Bioelectron
; 175: 112864, 2021 Mar 01.
Article
en En
| MEDLINE
| ID: mdl-33309217
We report the design of robust sensors for measuring intracellular pH, based on the native DNA i-motifs (iMs) found in neurodegeneration- or carcinogenesis-related genes. Those iMs appear to be genomic regulatory elements and might modulate transcription in response to pH stimuli. Given their intrinsic sensitivity to minor pH changes within the physiological range, such noncanonical DNA structures can be used as sensor core elements without additional modules other than fluorescent labels or quenchers. We focused on several iMs that exhibited fast folding/unfolding kinetics. Using stopped-flow techniques and FRET-melting/annealing assays, we confirmed that the rates of temperature-driven iM-ssDNA transitions correlate with the rates of the pH-driven transitions. Thus, we propose FRET-based hysteresis analysis as an express method for selecting sensors with desired kinetic characteristics. For the leading fast-response sensor, we optimized the labelling scheme and performed intracellular calibration. Unlike the commonly used small-molecule pH indicators, that sensor was transferred efficiently to cell nuclei. Considering its favourable kinetic characteristics, the sensor can be used for monitoring proton dynamics in the nucleus. These results argue that the 'genome-inspired' design is a productive approach to the development of biocompatible molecular tools.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Técnicas Biosensibles
Idioma:
En
Revista:
Biosens Bioelectron
Asunto de la revista:
BIOTECNOLOGIA
Año:
2021
Tipo del documento:
Article
País de afiliación:
Rusia
Pais de publicación:
Reino Unido