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Biology subjects

Hegge, J.

Publications and source records attributed to Hegge, J..

2 recordsLinked to original sources

High-Speed Super-Resolution Imaging Using Protein-Assisted DNA-PAINT

Super-resolution imaging allows for visualization of cellular structures on a nanoscale level. DNA-PAINT (DNA Point Accumulation In Nanoscale Topology) is a super-resolution method that depends on the binding and unbinding of DNA imager strands. The current DNA-PAINT technique suffers from slow acquisition due to the low binding rate of the imager strands. Here we report on a method where imager strands are loaded into a protein, Argonaute (Ago), that allows for faster binding. Ago pre-orders the DNA imager strand into a helical conformation, allowing for 10 times faster target binding. Using a 2D DNA origami structure, we demonstrate that Ago-assisted DNA-PAINT (Ago-PAINT) can speed up the current DNA-PAINT technique by an order of magnitude while maintaining the high spatial resolution. We envision this tool to be useful not only for super-resolution imaging, but also for other techniques that rely on nucleic-acid interactions.

biophysics↗

Argonaute bypasses cellular obstacles without hindrance during target search

Argonaute (Ago) proteins are key players in gene regulation in eukaryotes and host defense in prokaryotes. For specific interference, Ago relies on base pairing between small nucleic acid guides and complementary target sequences. To efficiently scan nucleic acid chains for potential targets, Ago must bypass both secondary structures in mRNA and single stranded DNA as well as protein barriers. Through single-molecule FRET, we reveal that lateral diffusion is mediated mainly through protein-nucleic acid interactions, rather than interactions between the guide and targeted strand. This allows Ago to scan for targets with high efficiency but without maintaining tight contact with the DNA backbone. Real-time observations show that Ago "glides" short distances over secondary structures while using intersegmental jumps to reduce scanning redundancy and bypass protein barriers. Our single-molecule method in combination with kinetic analysis may serve as a novel platform to study the effect of sequence on search kinetics for other nucleic acid-guided proteins.

biophysics↗