Monitoring G-Quadruplex Formation with DNA Carriers and Solid-State Nanopores

Abstract

G-quadruplexes (Gq) are guanine-rich DNA structures formed by single-stranded DNA. They are of paramount significance to gene expression regulation, but also drug targets for cancer and human viruses. Current ensemble and single-molecule methods require fluorescent labels, which can affect Gq folding kinetics. Here we introduce, a single-molecule Gq nanopore assay (smGNA) to detect Gqs and kinetics of Gq formation. We use ~5 nm solid-state nanopores to detect various Gq structural variants attached to designed DNA carriers. Gqs can be identified by localizing their positions along designed DNA carriers establishing smGNA as a tool for Gq mapping. In addition, smGNA allows for discrimination of (un-)folded Gq structures, provides insights into singlemolecule kinetics of G-quadruplex folding, and probes quadruplex-to-duplex structural transitions. smGNA can elucidate the formation of G-quadruplexes at the single-molecule level without labelling and has potential implications on the study of these structures both in single-stranded DNA and in genomic samples..

DNA Structural Barcode Copying and Random Access

“…As a proof‐of‐concept, we used DNA dumbbells (double DNA hairpins). However, a DNA protrusion can be replaced with any nanopore signal‐inducing secondary structures such us DNA hairpins, [ 8 ] G‐quadruplexes, [ 23 ] or a non‐complementary DNA loop that contains a binding site for additional DNA nanostructures [ 24 ] or biotinylated strands. [ 13 ] In addition, the binding site can be in the loop region of the DNA stem‐loop structure and both structures could be copied and accessed in a similar way as DNA dumbbells and serve as additional ways to create DNA structural barcodes.…”

“…As a proof-of-concept, we used DNA dumbbells (double DNA hairpins). However, a DNA protrusion can be replaced with any nanopore signal-inducing secondary structures such us DNA hairpins, [8] G-quadruplexes, [23] or a non-complementary DNA loop that contains a binding site for additional DNA nanostructures [24] or biotinylated strands. [13] In addition, the A B C Figure 4.…”

Label-free single-molecule identification of telomere G-quadruplexes with a solid-state nanopore sensor

“…We analyzed the current blockage in the rst tens of seconds, as many works have proved that the time scale of the G4 folding process is of a few seconds or even microseconds. 45,51 10 nM DNA1, DNA2 and long-sequence DNA6 were subjected to the SSN system with a pore size of 3.5 nm (for DNA1 and DNA2) and 4.2 nm (for DNA6) in both 1 M K + and Na + for folding/unfolding process investigation. Fig.…”

“…For instance, Long's group has utilized silicon nitride nanopores for ready determination of i-motif structure as well as DNA dynamics; [37][38][39] the employment of tiny SSN for sieving out G4 structure was also reported; 40,41 Jiang's group have observed the G4 DNA conformational switching in a responsive nanochannel; 42,43 imotif folding dynamics 44 and G4 formation with DNA carrier in SSN was also reported. 45 However, inter-and intramolecular and consecutive G4 have not been detected with SSN since interand intramolecular DNA with distinct base permutation and stability may perform differently in organisms, and consecutive G4 units are the dominating factor for diseases.…”

Discrimination of RNA fiber structures using solid-state nanopores

“…Solid-state nanopores have also been used to discriminate between chromatin sub-structures such as histone monomers, tetramers, and octamers. 20 Nanopores have been used to perform force spectroscopy of histone-DNA interactions in individual nucleosomes, 21 protein-DNA interactions, 22,23 study DNA modifications, 24 study folded, 25 decorated molecules 26 and map DNA sequence. 17 Here, we used solid-state nanopores (Fig.…”