Non-equilibrium structural dynamics of supercoiled DNA plasmids exhibits asymmetrical relaxation

Shaheen, Cynthia; Hastie, Cameron; Metera, Kimberly; Scott, Shane; Zhang, Zhi; Chen, Sitong; Gu, Gracia; Weber, Lisa; Munsky, Brian; Kouzine, Fedor; Levens, David; Benham, Craig J.; Leslie, Sabrina

doi:10.1093/nar/gkac101

Cited by 5 publications

(5 citation statements)

References 39 publications

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“…FRET data were analyzed using custom analysis software developed in MATLAB. Initially, the positions of individual microwells were mapped, following the approach detailed in a previous report by Shaheen et al 37 Subsequently, average intensities per microwell per imaging frame were calculated and normalized, as described in the Supporting Information.…”

Section: Methodsmentioning

confidence: 99%

Quantitative Visualization of Lipid Nanoparticle Fusion as a Function of Formulation and Process Parameters

Kamanzi,

Zhang,

et al. 2024

ACS Nano

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Lipid nanoparticles (LNPs) have proven to be promising delivery vehicles for RNA-based vaccines and therapeutics, particularly in LNP formulations containing ionizable cationic lipids that undergo protonation/deprotonation in response to buffer pH changes. These nanoparticles are typically formulated using a rapid mixing technique at low pH, followed by a return to physiological pH that triggers LNP− LNP fusion. A detailed understanding of these dynamic processes is crucial to optimize the overall performance and efficiency of LNPs. However, knowledge gaps persist regarding how particle formation mechanisms impact drug loading and delivery functions. In this work, we employ single-molecule Convex Lens-induced Confinement (CLiC) microscopy in combination with Forster resonance energy transfer (FRET) measurements to study LNP fusion dynamics in relation to various formulation parameters, including lipid concentration, buffer conditions, drug loading ratio, PEG-lipid concentrations, and ionizable lipid selection. Our results reveal a strong correlation between the measured fusion dynamics and the formulation parameters used; these findings are consistent with DLS and Cryo-TEM-based assays. These measurements offer a cost-effective method for characterizing and screening potential drug candidates and can provide additional insights into their design, with opportunities for optimization.

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Section: Methodsmentioning

confidence: 99%

Quantitative Visualization of Lipid Nanoparticle Fusion as a Function of Formulation and Process Parameters

Kamanzi,

Zhang,

et al. 2024

ACS Nano

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“…Much remains to be done before dynamical models can be designed and implemented that are capable of handling competing transitions in kilobase scale domains with any base sequence in complex, evolving environments. But on the experimental side, intriguing new strategies are elucidating both equilibrium and dynamical aspects of superhelical DNA structural transitions ( 96 , 119 ).…”

Section: Limitations and Opportunitiesmentioning

confidence: 99%

DNA superhelicity

Benham

2023

Nucleic Acids Research

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Closing each strand of a DNA duplex upon itself fixes its linking number L. This topological condition couples together the secondary and tertiary structures of the resulting ccDNA topoisomer, a constraint that is not present in otherwise identical nicked or linear DNAs. Fixing L has a range of structural, energetic and functional consequences. Here we consider how L having different integer values (that is, different superhelicities) affects ccDNA molecules. The approaches used are primarily theoretical, and are developed from a historical perspective. In brief, processes that either relax or increase superhelicity, or repartition what is there, may either release or require free energy. The energies involved can be substantial, sufficient to influence many events, directly or indirectly. Here two examples are developed. The changes of unconstrained superhelicity that occur during nucleosome attachment and release are examined. And a simple theoretical model of superhelically driven DNA structural transitions is described that calculates equilibrium distributions for populations of identical topoisomers. This model is used to examine how these distributions change with superhelicity and other factors, and applied to analyze several situations of biological interest.

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“…This was demonstrated by changing the ionic strength of the solution, which strongly affects DNA stretching, and further by adding cationic surfactants that condense DNA and restriction enzymes that digest DNA (Henkin et al, 2016). CLiC has in more recent years been implemented in combination with other nanoscale features, such as nanopit arrays, allowing for other types of studies on DNA, in particular supercoiled DNA (Scott et al, 2018(Scott et al, , 2019Shaheen et al, 2022).…”

Section: Clic (Convex Lens-induced Confinement)mentioning

confidence: 99%

DNA in nanochannels: theory and applications

Frykholm

Müller

et al. 2022

Quart. Rev. Biophys.

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Nanofluidic structures have over the last two decades emerged as a powerful platform for detailed analysis of DNA on the kilobase pair length scale. When DNA is confined to a nanochannel, the combination of excluded volume and DNA stiffness leads to the DNA being stretched to near its full contour length. Importantly, this stretching takes place at equilibrium, without any chemical modifications to the DNA. As a result, any DNA can be analyzed, such as DNA extracted from cells or circular DNA, and it is straight-forward to study reactions on the ends of linear DNA. In this comprehensive review, we first give a thorough description of the current understanding of the polymer physics of DNA and how that leads to stretching in nanochannels. We then describe how the versatility of nanofabrication can be used to design devices specifically tailored for the problem at hand, either by controlling the degree of confinement or enabling facile exchange of reagents to measure DNA-protein reaction kinetics. The remainder of the review focuses on two important applications of confining DNA in nanochannels. The first is optical DNA mapping, which provides the genomic sequence of intact DNA molecules in excess of 100 kilobase pairs in size, with kilobase pair resolution, through labeling strategies that are suitable for fluorescence microscopy. In this section, we highlight solutions to the technical aspects of genomic mapping, including the use of enzyme-based labeling and affinitybased labeling to produce the genomic maps, rather than recent applications in human genetics. The second is DNA-protein interactions, and several recent examples of such studies on DNA compaction, filamentous protein complexes, and reactions with DNA ends are presented. Taken together, these two applications demonstrate the power of DNA confinement and nanofluidics in genomics, molecular biology, and biophysics.

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Non-equilibrium structural dynamics of supercoiled DNA plasmids exhibits asymmetrical relaxation

Cited by 5 publications

References 39 publications

Quantitative Visualization of Lipid Nanoparticle Fusion as a Function of Formulation and Process Parameters

Quantitative Visualization of Lipid Nanoparticle Fusion as a Function of Formulation and Process Parameters

DNA superhelicity

DNA in nanochannels: theory and applications

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