Duplex DNA Is Weakened in Nanoconfinement

Abstract

For proteins and DNA secondary structures such as G-quadruplexes and i-motifs, nanoconfinement can facilitate their folding and increase structural stabilities. However, the properties of the physiologically prevalent B-DNA duplex have not been elucidated inside the nanocavity. Using a 17-bp DNA duplex in the form of a hairpin stem, here, we probed folding and unfolding transitions of the hairpin DNA duplex inside a DNA origami nanocavity. Compared to the free solution, the DNA hairpin inside the nanocage with a 15 × 15 nm cross section showed a drastic decrease in mechanical (20 → 9 pN) and thermodynamic (25 → 6 kcal/mol) stabilities. Free energy profiles revealed that the activation energy of unzipping the hairpin DNA duplex decreased dramatically (28 → 8 kcal/mol), whereas the transition state moved closer to the unfolded state inside the nanocage. All of these indicate that nanoconfinement weakens the stability of the hairpin DNA duplex to an unexpected extent. In a DNA hairpin made of a stem that contains complementary telomeric G-quadruplex (GQ) and i-motif (iM) forming sequences, formation of the Hoogsteen base pairs underlining the GQ or iM is preferred over the Watson−Crick base pairs in the DNA hairpin. These results shed light on the behavior of DNA in nanochannels, nanopores, or nanopockets of various natural or synthetic machineries. It also elucidates an alternative pathway to populate noncanonical DNA over B-DNA in the cellular environment where the nanocavity is abundant.

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

“…To address the formation of G4s in double-stranded DNA, several studies have used G4-forming sequences embedded in doublestranded DNA [56,88,89] or long DNA hairpins [90] (Figure 2D,E). Together with the DNA origami technique, the constructed DNA tether in force spectroscopy can contain a G4 structure placed inside a DNA origami nanocage that allows quantification of the folding kinetics of G4s in a confined space [91][92][93]. (Adapted with permission from [50], American Chemical Society; [61], Oxford Academic; [87], American Chemical Society).…”

“…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 ].…”

Chiral transfer‐dictated self‐assembly of chiral block copolymers

“…[23,257] Also, the confining effects imposed significant impacts to their physical properties and functionalities. [258,259] For example, a tobacco mosaic virus contained a single RNA chain, associated with 2130 identical proteins. The formation of these proteins involved a series folding of the peptide chains, which displayed a disk-like feature with a dimension correlated with two turns of the RNA helix.…”

Structure of a DNA G-Quadruplex Related to Osteoporosis with a G-A Bulge Forming a Pseudo-loop

“…G-rich sequences able to fold into G-quadruplex structures are not randomly dispersed throughout the genome, but enriched within regions associated with gene regulation, including promoters, introns and UTRs, and at the ends of the chromosomes, where they play an important role in telomere biology [ 13 , 14 , 16 , 17 , 18 , 19 , 20 ]. In specific localized environments the formation of non-canonical DNA structures can even be preferred over B DNA form [ 21 ].…”