Evaluating the effect of ionic strength on PNA:DNA duplex formation kinetics

Swenson, Colin; Lackey, Hershel H.; Reece, Eric J.; Harris, Joel M.; Heemstra, Jennifer M.; Peterson, Eric M.

doi:10.1039/d1cb00025j

Cited by 13 publications

(15 citation statements)

References 55 publications

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“…The magnitude of the observed k a rates is consistent with that observed by a wide range of kinetic studies performed with short nucleotides. 29,40,41,43,47 Together, the decrease in k d and increase in k a for the OPNA-modified strands led to an ∼10− 30 fold increase in affinity compared to the OPNA-0 PNA control, Figure 3A.…”

Section: ■ Materials and Methodsmentioning

confidence: 96%

“…The thermal effects on k a and k d are consistent with other DNA−DNA and PNA-DNA hybridization systems. 40,41 The equilibrium dissociation constants (K d ) calculated from the SPR hybridization rate constants for experiments performed at different temperatures are presented in Table 3 for each of the OPNA. As seen for OPNA-1A the affinity decrease with increasing temperature from 20 to 30 °C (1.2 to 11 nM, respectively) is consistent primarily with the fold increase in the dissociation rate, k d .…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

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Peptide Nucleic Acids Containing Cationic/Amino-Alkyl Modified Bases Promote Enhanced Hybridization Kinetics and Thermodynamics with Single-Strand DNA

Podlaski,

Cornwell,

Wong

et al. 2023

ACS Omega

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Peptide nucleic acids (PNAs) are antisense molecules with excellent polynucleotide hybridization properties; they are resistant to nuclease degradation but often have poor cell permeability leading to moderate cellular activity and limited clinical results. The addition of cationic substitutions (positive charges) to PNA molecules greatly increases cell permeability. In this report, we describe the synthesis and polynucleotide hybridization properties of a novel cationic/amino-alkyl nucleotide base-modified PNA (OPNA). This study was designed to quantitate the effect the cationic/amino-alkyl nucleotide base modification had on the kinetic and thermodynamic properties of OPNA-DNA hybridization using surface plasmon resonance and UV thermal melt studies. Kinetic studies reveal a favorable 10−30 fold increase in affinity for a single cationic modification on the base of an adenine, cytosine, or guanidine OPNA sequence compared to the nonmodified PNA strand. The increase in affinity is correlated directly with a favorable decrease in the dissociation rate constant and increase in the association rate constant. Introducing additional amino-alkyl base modifications further favors a decrease in the dissociation rate (3−10-fold per amino-alkyl). The thermodynamics driving the OPNA hybridization is promoted by an additional favorable −80 kJ/mol enthalpy of binding for a single amino-alkyl modification compared to the PNA strand. This increase in enthalpy is consistent with an ion−ion interaction with the DNA strand. These kinetic and thermodynamic hybridization studies reveal for the first time that this type of cationic/amino-alkyl base-modified PNA has favorable hybridization properties suitable for development as an antisense oligomer.

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Section: ■ Materials and Methodsmentioning

confidence: 96%

Section: ■ Materials and Methodsmentioning

confidence: 99%

Peptide Nucleic Acids Containing Cationic/Amino-Alkyl Modified Bases Promote Enhanced Hybridization Kinetics and Thermodynamics with Single-Strand DNA

Podlaski,

Cornwell,

Wong

et al. 2023

ACS Omega

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“…For instance, presence of metal cations can reduce the electrostatic repulsion between the phosphate groups of DNA fragments, thus inducing their condensation. [44,56] Furthermore, hydrophilic neutral polymer (e.g., polyethylene glycol (PEG)) in the DNA solution can cause phase exclusion of DNA fragments (known as "Ψ-condensation"). [47,57,58] However, temperature of the DNA solution does not affect the critical concentration, confirming the entropic-driven phase transition.…”

Section: Lcs From Dnamentioning

confidence: 99%

Liquid Crystalline Systems from Nature and Interaction of Living Organisms with Liquid Crystals

Kim

et al. 2022

Advanced Materials

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The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/adma.202204275. of biosensors with additional functionalities (e.g., self-regulated drug release) that are not available in previous systems is reviewed. Examples addressed in this review convey the message that the intersection between biomaterials and LCs offers deep insights into fundamental understanding of biomaterials, and provides resources for development of transformative technologies.

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“…The goal is now to maximize the residence time in the FRET regime, which can be achieved by tuning the dye binding and dissociation rates, plus the sampling rate of the measurement – all while considering the dye-specific bleach rate. A Monte Carlo simulation based on experiment-derived rate constants [37,41–43] illustrates the situation ( Fig. 2C ).…”

Section: Breaking the Photobleaching Limit In Time-resolved Smfret Wi...mentioning

confidence: 99%

“…3i ) are an obvious choice borrowed from super-resolution imaging [37]: an ssDNA oligo may be covalently attached to a protein of interest as a docking target for a complementary dye-cycler oligo. A wealth of information is available on hybridization kinetics [37,41,43,46]. The hybridization rate depends on the cycler’s diffusion time, and is tunable with cycler concentration, temperature, and salt concentration, whereas the dissociation rate can be tuned via the DNA duplex length and sequence (GC content, mismatches [41,47]), salt conditions, and temperature [41].…”

Section: Breaking the Photobleaching Limit In Time-resolved Smfret Wi...mentioning

confidence: 99%

Can DyeCycling break the photobleaching limit in single-molecule FRET?

Vermeer

Schmid

2022

Preprint

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Biomolecular systems, such as proteins, crucially rely on dynamic processes at the nanoscale. Detecting biomolecular nanodynamics is therefore key to obtaining a mechanistic understanding of the energies and molecular driving forces that control biomolecular systems. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful technique to observe in real-time how a single biomolecule proceeds through its functional cycle involving a sequence of distinct structural states. Currently, this technique is fundamentally limited by irreversible photobleaching, causing the untimely end of the experiment and thus, a prohibitively narrow temporal bandwidth of ≤ 3 orders of magnitude. Here, we introduce ″DyeCycling″, a measurement scheme with which we aim to break the photobleaching limit in single-molecule FRET. We introduce the concept of spontaneous dye replacement by simulations, and as an experimental proof-of-concept, we demonstrate the intermittent observation of a single biomolecule for one hour with a time resolution of milliseconds. Theoretically, DyeCycling can provide >100-fold more information per single molecule than conventional smFRET. We discuss the experimental implementation of DyeCycling, its current and fundamental limitations, and specific biological use cases. Given its general simplicity and versatility, DyeCycling has the potential to revolutionize the field of time-resolved smFRET, where it may serve to unravel a wealth of biomolecular dynamics by bridging from milliseconds to the hour range.

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Evaluating the effect of ionic strength on PNA:DNA duplex formation kinetics

Abstract: Peptide nucleic acid (PNA) is a unique synthetic nucleic acid analog that has been adopted for use in many biological applications. These applications rely upon the robust Franklin-Watson-Crick base pairing...

Cited by 13 publications

References 55 publications

Peptide Nucleic Acids Containing Cationic/Amino-Alkyl Modified Bases Promote Enhanced Hybridization Kinetics and Thermodynamics with Single-Strand DNA

Peptide Nucleic Acids Containing Cationic/Amino-Alkyl Modified Bases Promote Enhanced Hybridization Kinetics and Thermodynamics with Single-Strand DNA

Liquid Crystalline Systems from Nature and Interaction of Living Organisms with Liquid Crystals

Can DyeCycling break the photobleaching limit in single-molecule FRET?

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