Pointwise prediction of protein diffusive properties using machine learning
Journal of Physics: Photonics IOP Publishing (2025)
Abstract:
Tunable fluorogenic DNA probes drive fast and high-resolution single-molecule fluorescence imaging
Nucleic Acids Research 53:13 (2025) gkaf593
Abstract:
A main limitation of single-molecule fluorescence (SMF) measurements is the 'high concentration barrier', describing the maximum concentration of fluorescent species tolerable for sufficient signal-to-noise ratio. To address this barrier in several SMF applications, we design fluorogenic probes based on short single-stranded DNAs, fluorescing only upon hybridizing to their complementary target sequence. We engineer the quenching efficiency and fluorescence enhancement upon duplex formation through screening several fluorophore-quencher combinations, label lengths, and sequence motifs, which we utilize as tuning screws to adapt our labels to different experimental designs. Using these fluorogenic probes, we can perform SMF experiments at concentrations of 10 μM fluorescent labels; this concentration is 100-fold higher than the operational limit for standard TIRF experiments. We demonstrate the ease of implementing these probes into existing protocols by performing super-resolution imaging with DNA-PAINT, employing a fluorogenic 6-nt-long imager; through the faster acquisition of binding events, the imaging of viral genome segments could be sped up significantly to achieve extraction of 20-nm structural features with only ∼150 s of imaging. The exceptional tunability of our probe design will overcome concentration barriers in SMF experiments and unlock new possibilities in super-resolution imaging, molecular tracking, and single-molecule fluorescence energy transfer (smFRET).Single-Molecule Imaging for Unraveling the Functional Diversity of 10–23 DNAzymes
Analytical Chemistry American Chemical Society (ACS) (2025)
In vivo single-molecule imaging of RecB reveals efficient repair of DNA damage in Escherichia coli
Nucleic Acids Research Oxford University Press 53:10 (2025) gkaf454
Abstract:
Efficient DNA repair is essential for maintaining genome integrity and ensuring cell survival. In Escherichia coli, RecBCD plays a crucial role in processing DNA ends, following a DNA double-strand break (DSB), to initiate repair. While RecBCD has been extensively studied in vitro, less is known about how it contributes to rapid and efficient repair in living bacteria. Here, we use single-molecule microscopy to investigate DNA repair in real time in E. coli. We quantify RecB single-molecule mobility and monitor the induction of the DNA damage response (SOS response) in individual cells. We show that RecB binding to DNA ends caused by endogenous processes leads to efficient repair without SOS induction. In contrast, repair is less efficient in the presence of exogenous damage or in a mutant strain with modified RecB activities, leading to high SOS induction. Our findings reveal how subtle alterations in RecB activity profoundly impact the efficiency of DNA repair in E. coli.Ribosome phenotypes for rapid classification of antibiotic-susceptible and resistant strains of Escherichia coli
Communications Biology Nature Research 8:1 (2025) 319