Kinetic Referencing Allows Identification of Epigenetic Cytosine Modifications by Single-Molecule Hybridization Kinetics and Superresolution DNA-PAINT Microscopy

Abstract

We develop a DNA origami-based internal kinetic referencing system with a colocalized reference and target molecule to provide increased sensitivity and robustness for transient binding kinetics. To showcase this, we investigate the subtle changes in binding strength of DNA oligonucleotide hybrids induced by cytosine modifications. These cytosine modifications, especially 5-methylcytosine but also its oxidized derivatives, have been increasingly studied in the context of epigenetics. Recently revealed correlations of epigenetic modifications and disease also render them interesting biomarkers for early diagnosis. Internal kinetic referencing allows us to probe and compare the influence of the different epigenetic cytosine modifications on the strengths of 7nucleotide long DNA hybrids with one or two modified nucleotides by single-molecule imaging of their transient binding, revealing subtle differences in binding times. Interestingly, the influence of epigenetic modifications depends on their position in the DNA strand, and in the case of two modifications, effects are additive. The sensitivity of the assay indicates its potential for the direct detection of epigenetic disease markers.

Cryosectioning-enabled super-resolution microscopy for studying nuclear architecture at the single protein level

“…Furthermore, secKoned samples offer the addiKonal advantage of being more accessible to the imaging buffer and, thus, binding of imager strands. The perpetual nature of repeated imager binding at controllable rates is a hallmark of DNA-PAINT and has led to a diverse range of quanKtaKve in vitro 12,[28][29][30][31] and in situ 11,28 applicaKons, such as the counKng of target molecules within fixed cells and Kssues via qPAINT 11 . A physically-secKoned sample that is openly accessible to the imaging soluKon could potenKally exhibit improved binding kineKcs, thus elevaKng our ability to leverage DNA-PAINT as a quanKtaKve tool.…”