Fast and multiplexed superresolution imaging with DNA-PAINT-ERS

Abstract

DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) facilitates multiplexing in superresolution microscopy but is practically limited by slow imaging speed. To address this issue, we propose the additions of ethylene carbonate (EC) to the imaging buffer, sequence repeats to the docking strand, and a spacer between the docking strand and the affinity agent. Collectively termed DNA-PAINT-ERS (E = EC, R = Repeating sequence, and S = Spacer), these strategies can be easily integrated into current DNA-PAINT workflows for both accelerated imaging speed and improved image quality through optimized DNA hybridization kinetics and efficiency. We demonstrate the general applicability of DNA-PAINT-ERS for fast, multiplexed superresolution imaging using previously validated oligonucleotide constructs with slight modifications.

Probing Transient DNA Conformation Changes with an Intercalative Fluorescent Excimer

“…When the DNA length is over 12 bp, the excimer emission decreased. According to previous results from the DNA‐PAINT technique, imager strands with lengths ranging from 8 to 12 bp exhibit a metastable hybridization state [13] . Here, we measured the binding constants between K21 and dsDNAs with lengths ranging from 4 bp to 30 bp (Supporting Information, Figure S5 and Table S2).…”

DNA hybridisation kinetics using single-molecule fluorescence imaging

“…Another way to affect hybridisation kinetics is by introducing ethylene carbonate (EC) an aprotic solvent thought to increase the solubility of the DNA bases [68]. Studied at the single-molecule level, in the presence of 0-15% EC, there is a five to tenfold decrease in k off in the transient binding of a 9-nucleotide probe.…”

Probing Transient DNA Conformation Changes with an Intercalative Fluorescent Excimer

“…According to previous results from the DNA-PAINT technique,i mager strands with lengths ranging from 8t o1 2bp exhibit am etastable hybridization state. [13] Here,w em easured the binding constants between K21 and dsDNAs with lengths ranging from 4bpt o3 0bp( Supporting Information, Figure S5 and Table S2). We found that compared to the shorter (4 bp) or longer (30 bp) ones,the dsDNAs of 7-12 bp show intermediate affinities towards K21, which further support the idea about DNAm etastable hybridization state Figure 3.…”