Accelerated Left-Handed DNA-PAINT Using Fluorogenic Probes

Area of Science:

• Biophysics
• Microscopy
• Molecular Biology

Background:

• Single-molecule localization microscopy (SMLM) achieves nanoscale resolution by localizing individual fluorescent molecules.
• DNA-mediated point accumulation for imaging in nanoscale topography (DNA-PAINT) is a powerful SMLM technique known for multiplexing and accuracy.

Purpose of the Study:

• To introduce a faster DNA-PAINT method utilizing left-handed DNA (L-DNA) probes.
• To reduce background noise and improve volumetric imaging capabilities in DNA-PAINT.

Main Methods:

• Development and application of left-handed DNA (L-DNA) probes for DNA-PAINT.
• Integration of fluorogenic probes with L-DNA-PAINT for quenched emission from free binders.
• Comparative analysis of binding kinetics, brightness, and localization precision between L-DNA and R-DNA probes.

Main Results:

• L-DNA probes significantly reduce background noise from unspecific binding.
• Fluorogenic L-DNA-PAINT enables volumetric imaging with quenched free binders.
• L-DNA-PAINT allows for shorter integration times and faster localization accumulation.
• Fluorogenic L-DNA and R-DNA probes exhibit indistinguishable binding kinetics, brightness, and precision.
• Absence of cross-reactivity between fluorogenic left- and right-handed probes expands multiplexing options.

Conclusions:

• L-DNA-PAINT offers a faster and lower-background alternative for super-resolution microscopy.
• The combination of L-DNA probes and fluorogenic designs greatly enhances DNA-PAINT's experimental versatility.
• This advancement facilitates reliable, high-resolution, multiplexed imaging in biological samples.