How fluorescent labels affect the kinetics of the toehold-mediated DNA strand displacement reaction

Abstract

The end modification using fluorophore dyes or quenchers to or close to the toehold domain can significantly modulate the kinetics of toehold-mediated strand displacement reaction (TMSDR) by almost two orders...

Strategies for Controlling the Spatial Orientation of Single Molecules Tethered on DNA Origami Templates Physisorbed on Glass Substrates: Intercalation and Stretching

“…In one application, the separation of two dyes with a distance resolution of ~0.04 nm was achieved using a hinged construct [ 30 ]. Although dyes can be precisely positioned on the DNA origami template under cryogenic temperature conditions [ 31 ], in the last few years the impact of the dye’s surrounding environment [ 32 ]—i.e., DNA breathing [ 33 , 34 ], DNA molecular structure [ 35 ], dye-DNA interactions [ 36 , 37 , 38 , 39 ], and linker type [ 40 ]—has been recognized as a crucial factor governing dye orientation relative to the DNA molecule and, consequently, affecting the performance of the particular applications [ 32 , 35 , 36 , 37 , 38 , 39 , 40 ]. All these efforts indicate that much remains to be learned about the DNA molecule [ 41 ], particularly when using it as a functional material for molecular organization, including orientational control.…”

Strong sequence dependence in RNA/DNA hybrid strand displacement kinetics

“…These toehold exchange reactions were monitored over time by assessing the concentration of released incumbent strand over the course of the reaction. We made use of a distinct fluorescent reporter detection system (Figure 3A) to assess the concentration of released incumbent to avoid directly labelling the strands within the reaction of interest, which may otherwise influence the kinetics of strand displacement (44). The reporter complex was composed of a fluorescently (AlexaFluor488)-labelled strand ( F ) which was partially complementary to a quencher (IowaBlack-FQ)-associated strand ( Q ).…”

Design strategies for countering the effect of fluorophore-quencher labelling on DNA hairpin thermodynamics

“…9–11 There is an inherent yet inaccurate assumption that the unmodified oligos and modified probes are equivalent as recently studied by Li et al . 12 In their study of toehold-mediated DNA strand displacement involving duplex substrates, the end modification of duplexes slows the strand displacement kinetics, while modification on the toehold domains accelerates the kinetics.…”

“…For example, the experimental feasibility of a DNA circuit design is often checked via gel electrophoresis using a high concentration of unmodified oligos before transiting to fluorescence measurement using a relatively lower concentration of the modified probes. [9][10][11] There is an inherent yet inaccurate assumption that the unmodified oligos and modified probes are equivalent as recently studied by Li et al 12 In their study of toehold-mediated DNA strand displacement involving duplex substrates, the end modification of duplexes slows the strand displacement kinetics, while modification on the toehold domains accelerates the kinetics.…”