Submolecular dissection reveals strong and specific binding of polyamide–pyridostatin conjugates to human telomere interface

Abstract

To modulate biological functions, G-quadruplexes in genome are often non-specifically targeted by small molecules. Here, specificity is increased by targeting both G-quadruplex and its flanking duplex DNA in a naturally occurring dsDNA–ssDNA telomere interface using polyamide (PA) and pyridostatin (PDS) conjugates (PA-PDS). We innovated a single-molecule assay in which dissociation constant ( K d ) of the conjugate can be separately evaluated from the binding of either PA or PDS. We found K d of 0.8 nM for PA-PDS, which is much lower than PDS ( K d ∼ 450 nM) or PA ( K d ∼ 35 nM). Functional assays further indicated that the PA-PDS conjugate stopped the replication of a DNA polymerase more efficiently than PA or PDS. Our results not only established a new method to dissect multivalent binding into actions of individual monovalent components, they also demonstrated a strong and specific G-quadruplex targeting strategy by conjugating highly specific duplex-binding molecules with potent quadruplex ligands.

Dissection of nanoconfinement and proximity effects on the binding events in DNA origami nanocavity

“…Dibenzocyclooctyne-N-hydroxysuccinimidyl ester was purchased from Sigma Aldrich. Azide modified pyridostatin was prepared as described in literature ( 13 ). The streptavidin or anti-digoxigenin coated polystyrene beads were purchased from Spherotech.…”

Mechanical Stability of DNA Corona Phase on Gold Nanospheres

“…We found that average mechanical stability of (dA) 21 strands adsorbed on these three AuNPs is ∼40 pN. This value is significantly higher than the stall force of DNA/RNA polymerases. − Therefore, similar to the G-quadruplex that may serve as a mechanical block , to DNA replication or RNA transcription processes, the AuNP-poly­(dA) may also serve similar functions, given that there are plenty of poly­(dA) segments in genomes of many species. − This potential function further expands the applicability of AuNP in biomedicines. This approach of determining the mechanical stability of DNA corona phase on nanoparticles can also be extended to other materials such as carbon nanotubes which are of growing interest in recent years .…”

“…This value is significantly higher than the stall force of DNA/RNA polymerases. 37−39 Therefore, similar to the G-quadruplex that may serve as a mechanical block 47,48 to DNA replication or RNA transcription processes, the AuNP-poly(dA) may also serve similar functions, given that there are plenty of poly(dA) segments in genomes of many species. 41−43 This potential function further expands the applicability of AuNP in biomedicines.…”

Characterization of G-Quadruplexes Folding/Unfolding Dynamics and Interactions with Proteins from Single-Molecule Force Spectroscopy

“…The binding of PDS to telomere G4s resulted in the shift of the unfolding force peak of G4s in K + solution from ~21 pN to~41 pN, suggesting a strong reduction in unfolding rate. Since then, the applications of force–spectroscopy techniques further enable the characterization of interactions between quadruplexes and quadruplexes [ 108 ], small ligands and quadruplexes [ 52 , 109 , 110 , 111 , 112 ], proteins and quadruplexes [ 63 , 64 , 65 ], and study on factors that affect the folding/unfolding dynamics of G4s, including molecular crowding [ 113 ], force at different directions [ 84 , 110 ], nanoconfinement [ 91 , 92 , 93 ], DNA superhelicity [ 88 ], and RNA transcripts [ 114 ]. The force spectroscopy has also been used to study the folding/unfolding dynamics of other four strands nucleic acid structures i-motif, which are located at the opposite strand of G4s [ 115 , 116 ], and the molecular switch between G4s and i-motif [ 117 ].…”