Measuring DNA Hybridization Kinetics in Live Cells Using a Time-Resolved 3D Single-Molecule Tracking Method

Chen, Yuan I.; Chang, Yin Jui; Nguyễn, Tùng Lâm; Liu, Cong; Phillion, Stephanie; Kuo, Yu An; Vu, Huong T.; Liu, Angela; Liu, Chia‐Jyi; Hong, Suk-In; Ren, Pengyu; Yankeelov, Thomas E.; Yeh, Hsin Chih

doi:10.1021/jacs.9b08036

Cited by 17 publications

(26 citation statements)

References 39 publications

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“…Exogenously delivered DNA has a cellular half-life on the order of minutes and is susceptible to unintended interactions with endogenous macromolecules ( 24 ), all of which adversely affect the performance of the device. While use of chemical modifications, such as 2′- O -methyl ribonucleotides ( 25 , 26 ), locked nucleic acids ( 25 ) and phosphorothioate linkages ( 26 ) can confer nuclease stability, they also alter duplex thermostability ( 27 ) and hybridization kinetics ( 28 , 29 ) in a unpredictable manner. Indeed, compared to native DNA, strand displacement reactions involving chemically modified nucleic acids are poorly characterized, making the design of corresponding devices extremely challenging ( 22 ).…”

Section: Introductionmentioning

confidence: 99%

Kinetics of heterochiral strand displacement from PNA–DNA heteroduplexes

Kundu

Young

Sczepanski

2021

Nucleic Acids Research

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Dynamic DNA nanodevices represent powerful tools for the interrogation and manipulation of biological systems. Yet, implementation remains challenging due to nuclease degradation and other cellular factors. Use of l-DNA, the nuclease resistant enantiomer of native d-DNA, provides a promising solution. On this basis, we recently developed a strand displacement methodology, referred to as ‘heterochiral’ strand displacement, that enables robust l-DNA nanodevices to be sequence-specifically interfaced with endogenous d-nucleic acids. However, the underlying reaction – strand displacement from PNA–DNA heteroduplexes – remains poorly characterized, limiting design capabilities. Herein, we characterize the kinetics of strand displacement from PNA–DNA heteroduplexes and show that reaction rates can be predictably tuned based on several common design parameters, including toehold length and mismatches. Moreover, we investigate the impact of nucleic acid stereochemistry on reaction kinetics and thermodynamics, revealing important insights into the biophysical mechanisms of heterochiral strand displacement. Importantly, we show that strand displacement from PNA–DNA heteroduplexes is compatible with RNA inputs, the most common nucleic acid target for intracellular applications. Overall, this work greatly improves the understanding of heterochiral strand displacement reactions and will be useful in the rational design and optimization of l-DNA nanodevices that operate at the interface with biology.

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Section: Introductionmentioning

confidence: 99%

Kinetics of heterochiral strand displacement from PNA–DNA heteroduplexes

Kundu

Young

Sczepanski

2021

Nucleic Acids Research

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“…To ensure quasi-robustness of the controller R b,g , we require that the desired input network R a fires at a slower timescale. For the interactions between the RNA species at a concentration of 30 molecules per cell, 0.1 reactions per second corresponds to a rate constant of the order of 10 6 M À1 s À1 , whereas the speed limit of nucleic-acid reactions inside cells is of the order of 10 8 M À1 s À1 [74]. If R a is a synthetic RNA-based network, the quasi-robust limit can be achieved by slowing down the input network via sequestration of the key domains in the secondary structures of the underlying species [17,54].…”

Section: Discussionmentioning

confidence: 99%

Quasi-robust control of biochemical reaction networks via stochastic morphing

Plesa

Stan

Ouldridge

et al. 2021

J. R. Soc. Interface.

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One of the main objectives of synthetic biology is the development of molecular controllers that can manipulate the dynamics of a given biochemical network that is at most partially known. When integrated into smaller compartments, such as living or synthetic cells, controllers have to be calibrated to factor in the intrinsic noise. In this context, biochemical controllers put forward in the literature have focused on manipulating the mean (first moment) and reducing the variance (second moment) of the target molecular species. However, many critical biochemical processes are realized via higher-order moments, particularly the number and configuration of the probability distribution modes (maxima). To bridge the gap, we put forward the stochastic morpher controller that can, under suitable timescale separations, morph the probability distribution of the target molecular species into a predefined form. The morphing can be performed at a lower-resolution, allowing one to achieve desired multi-modality/multi-stability, and at a higher-resolution, allowing one to achieve arbitrary probability distributions. Properties of the controller, such as robustness and convergence, are rigorously established, and demonstrated on various examples. Also proposed is a blueprint for an experimental implementation of stochastic morpher.

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“…We have recently used this HMM approach to characterize the hybridization kinetics of single diffusing DNA strands in solution 5 and in live cells. 6 In HMM approaches, a statistical model defines an expected distribution of measurement values based on a set of parameters, such as the means and standard deviations of Gaussian peaks representing the signal values associated with each state, and the transition probabilities between states. The ebFRET method characterizes the kinetics of FRET pairs based on FRET efficiency which is defined as EFRET = IA/(ID+IA), where IA and ID are the emission intensity of the acceptor and donor fluorophores, respectively.…”

Section: Supplementary Note S4 | Bayesian Methods To Measure Dna Bendimentioning

confidence: 99%

Three-Dimensional Two-Color Dual-Particle Tracking Microscope for Monitoring DNA Conformational Changes and Nanoparticle Landings on Live Cells

et al. 2020

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Here, we present a three-dimensional two-color dual-particle tracking (3D-2C-DPT) technique that can simultaneously localize two spectrally distinct targets in three dimensions with a time resolution down to 5 ms. The dual-targets can be tracked with separation distances from 33 to 250 nm with tracking precisions of ∼15 nm (for static targets) and ∼35 nm (for freely diffusing targets). Since each target is individually localized, a wealth of data can be extracted, such as the relative 3D position, the 2D rotation, and the separation distance between the two targets. Using this technique, we turn a double-stranded DNA (dsDNA)-linked dumbbell-like dimer into a nanoscopic optical ruler to quantify the bending dynamics of nicked or gapped dsDNA molecules in free solution by manipulating the design of dsDNA linkers (1-nick, 3-nt, 6-nt, or 9-nt single-strand gap), and the results show the increase of k on (linear to bent) from 3.2 to 10.7 s–1. The 3D-2C-DPT is then applied to observe translational and rotational motions of the landing of an antibody-conjugated nanoparticle on the plasma membrane of living cells, revealing the reduction of rotations possibly due to interactions with membrane receptors. This study demonstrates that this 3D-2C-DPT technique is a new tool to shed light on the conformational changes of biomolecules and the intermolecular interactions on plasma membrane.

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Measuring DNA Hybridization Kinetics in Live Cells Using a Time-Resolved 3D Single-Molecule Tracking Method

Cited by 17 publications

References 39 publications

Kinetics of heterochiral strand displacement from PNA–DNA heteroduplexes

Kinetics of heterochiral strand displacement from PNA–DNA heteroduplexes

Quasi-robust control of biochemical reaction networks via stochastic morphing

Three-Dimensional Two-Color Dual-Particle Tracking Microscope for Monitoring DNA Conformational Changes and Nanoparticle Landings on Live Cells

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