Single-Molecule Kinetics Reveal Cation-Promoted DNA Duplex Formation Through Ordering of Single-Stranded Helices

In this work, the kinetics of short, fully complementary oligonucleotides are investigated at the single-molecule level. Constructs 6–9 bp in length exhibit single exponential kinetics over 2 orders of magnitude time for both forward (kon, association) and reverse (koff, dissociation) processes. Bim...

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Published inBiophysical journal Vol. 105; no. 3; pp. 756 - 766
Main Authors Dupuis, Nicholas F., Holmstrom, Erik D., Nesbitt, David J.
Format Journal Article
LanguageEnglish
Published United States Elsevier Inc 06.08.2013
Biophysical Society
The Biophysical Society
Subjects
Online AccessGet full text
ISSN0006-3495
1542-0086
1542-0086
DOI10.1016/j.bpj.2013.05.061

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Abstract In this work, the kinetics of short, fully complementary oligonucleotides are investigated at the single-molecule level. Constructs 6–9 bp in length exhibit single exponential kinetics over 2 orders of magnitude time for both forward (kon, association) and reverse (koff, dissociation) processes. Bimolecular rate constants for association are weakly sensitive to the number of basepairs in the duplex, with a 2.5-fold increase between 9 bp (k′on = 2.1(1) × 106 M−1 s−1) and 6 bp (k′on = 5.0(1) × 106 M−1 s−1) sequences. In sharp contrast, however, dissociation rate constants prove to be exponentially sensitive to sequence length, varying by nearly 600-fold over the same 9 bp (koff = 0.024 s−1) to 6 bp (koff = 14 s−1) range. The 8 bp sequence is explored in more detail, and the NaCl dependence of kon and koff is measured. Interestingly, konincreases by >40-fold (kon = 0.10(1) s−1 to 4.0(4) s−1 between [NaCl] = 25 mM and 1 M), whereas in contrast, koffdecreases by fourfold (0.72(3) s−1 to 0.17(7) s−1) over the same range of conditions. Thus, the equilibrium constant (Keq) increases by ≈160, largely due to changes in the association rate, kon. Finally, temperature-dependent measurements reveal that increased [NaCl] reduces the overall exothermicity (ΔΔH° > 0) of duplex formation, albeit by an amount smaller than the reduction in entropic penalty (−TΔΔS° < 0). This reduced entropic cost is attributed to a cation-facilitated preordering of the two single-stranded species, which lowers the association free-energy barrier and in turn accelerates the rate of duplex formation.
AbstractList In this work, the kinetics of short, fully complementary oligonucleotides are investigated at the single-molecule level. Constructs 6-9 bp in length exhibit single exponential kinetics over 2 orders of magnitude time for both forward (kon, association) and reverse (koff, dissociation) processes. Bimolecular rate constants for association are weakly sensitive to the number of basepairs in the duplex, with a 2.5-fold increase between 9 bp (k'on = 2.1(1) 106 M-1 s-1) and 6 bp (k'on = 5.0(1) 106 M-1 s-1) sequences. In sharp contrast, however, dissociation rate constants prove to be exponentially sensitive to sequence length, varying by nearly 600-fold over the same 9 bp (koff = 0.024 s-1) to 6 bp (koff = 14 s-1) range. The 8 bp sequence is explored in more detail, and the NaCl dependence of kon and koff is measured. Interestingly, konincreases by >40-fold (kon = 0.10(1) s-1 to 4.0(4) s-1 between [NaCl] = 25 mM and 1 M), whereas in contrast, koffdecreases by fourfold (0.72(3) s-1 to 0.17(7) s-1) over the same range of conditions. Thus, the equilibrium constant (Keq) increases by approximately 160, largely due to changes in the association rate, kon. Finally, temperature-dependent measurements reveal that increased [NaCl] reduces the overall exothermicity ( Delta Delta H degree > 0) of duplex formation, albeit by an amount smaller than the reduction in entropic penalty (-T Delta Delta S degree < 0). This reduced entropic cost is attributed to a cation-facilitated preordering of the two single-stranded species, which lowers the association free-energy barrier and in turn accelerates the rate of duplex formation.
In this work, the kinetics of short, fully complementary oligonucleotides are investigated at the single-molecule level. Constructs 6-9 bp in length exhibit single exponential kinetics over 2 orders of magnitude time for both forward (k..., association) and reverse (koff, dissociation) processes. Bimolecular rate constants for association are weakly sensitive to the number of basepairs in the duplex, with a 2.5-fold increase between 9 bp (k'... = 2.1(1) x 10... M... s...) and 6 bp (k'... = 5.0(1) x 10... M... s...) sequences. In sharp contrast, however, dissociation rate constants prove to be exponentially sensitive to sequence length, varying by nearly 600-fold over the same 9 bp (k... = 0.024 s...) to 6 bp (koff = 14 s...) range. The 8 bp sequence is explored in more detail, and the NaCl dependence of kon and koff is measured. Interestingly, k... increases by >40-fold (kon = 0.10(1) s... to 4.0(4) s... between [NaCl] = 25 mM and 1 M), whereas in contrast, koffdecreases by fourfold (0.72(3) s... to 0.17(7) s... over the same range of conditions. Thus, the equilibrium constant (Keq) increases by ...160, largely due to changes in the association rate, kon. Finally, temperature-dependent measurements reveal that increased [NaCl] reduces the overall exothermicity (...H... > 0) of duplex formation, albeit by an amount smaller than the reduction in entropic penalty (-T...S... < 0). This reduced entropic cost is attributed to a cation-facilitated preordering of the two single-stranded species, which lowers the association free-energy barrier and in turn accelerates the rate of duplex formation. (ProQuest: ... denotes formulae/symbols omitted.)
In this work, the kinetics of short, fully complementary oligonucleotides are investigated at the single-molecule level. Constructs 6–9 bp in length exhibit single exponential kinetics over 2 orders of magnitude time for both forward (kₒₙ, association) and reverse (kₒff, dissociation) processes. Bimolecular rate constants for association are weakly sensitive to the number of basepairs in the duplex, with a 2.5-fold increase between 9 bp (k′ₒₙ = 2.1(1) × 10⁶ M⁻¹ s⁻¹) and 6 bp (k′ₒₙ = 5.0(1) × 10⁶ M⁻¹ s⁻¹) sequences. In sharp contrast, however, dissociation rate constants prove to be exponentially sensitive to sequence length, varying by nearly 600-fold over the same 9 bp (kₒff = 0.024 s⁻¹) to 6 bp (kₒff = 14 s⁻¹) range. The 8 bp sequence is explored in more detail, and the NaCl dependence of kₒₙ and kₒff is measured. Interestingly, kₒₙincreases by >40-fold (kₒₙ = 0.10(1) s⁻¹ to 4.0(4) s⁻¹ between [NaCl] = 25 mM and 1 M), whereas in contrast, kₒffdecreases by fourfold (0.72(3) s⁻¹ to 0.17(7) s⁻¹) over the same range of conditions. Thus, the equilibrium constant (Kₑq) increases by ≈160, largely due to changes in the association rate, kₒₙ. Finally, temperature-dependent measurements reveal that increased [NaCl] reduces the overall exothermicity (ΔΔH° > 0) of duplex formation, albeit by an amount smaller than the reduction in entropic penalty (−TΔΔS° < 0). This reduced entropic cost is attributed to a cation-facilitated preordering of the two single-stranded species, which lowers the association free-energy barrier and in turn accelerates the rate of duplex formation.
In this work, the kinetics of short, fully complementary oligonucleotides are investigated at the single-molecule level. Constructs 6-9 bp in length exhibit single exponential kinetics over 2 orders of magnitude time for both forward (kon, association) and reverse (koff, dissociation) processes. Bimolecular rate constants for association are weakly sensitive to the number of basepairs in the duplex, with a 2.5-fold increase between 9 bp (k'on = 2.1(1) × 10(6) M(-1) s(-1)) and 6 bp (k'on = 5.0(1) × 10(6) M(-1) s(-1)) sequences. In sharp contrast, however, dissociation rate constants prove to be exponentially sensitive to sequence length, varying by nearly 600-fold over the same 9 bp (koff = 0.024 s(-1)) to 6 bp (koff = 14 s(-1)) range. The 8 bp sequence is explored in more detail, and the NaCl dependence of kon and koff is measured. Interestingly, kon increases by >40-fold (kon = 0.10(1) s(-1) to 4.0(4) s(-1) between [NaCl] = 25 mM and 1 M), whereas in contrast, koff decreases by fourfold (0.72(3) s(-1) to 0.17(7) s(-1)) over the same range of conditions. Thus, the equilibrium constant (Keq) increases by ≈160, largely due to changes in the association rate, kon. Finally, temperature-dependent measurements reveal that increased [NaCl] reduces the overall exothermicity (ΔΔH° > 0) of duplex formation, albeit by an amount smaller than the reduction in entropic penalty (-TΔΔS° < 0). This reduced entropic cost is attributed to a cation-facilitated preordering of the two single-stranded species, which lowers the association free-energy barrier and in turn accelerates the rate of duplex formation.In this work, the kinetics of short, fully complementary oligonucleotides are investigated at the single-molecule level. Constructs 6-9 bp in length exhibit single exponential kinetics over 2 orders of magnitude time for both forward (kon, association) and reverse (koff, dissociation) processes. Bimolecular rate constants for association are weakly sensitive to the number of basepairs in the duplex, with a 2.5-fold increase between 9 bp (k'on = 2.1(1) × 10(6) M(-1) s(-1)) and 6 bp (k'on = 5.0(1) × 10(6) M(-1) s(-1)) sequences. In sharp contrast, however, dissociation rate constants prove to be exponentially sensitive to sequence length, varying by nearly 600-fold over the same 9 bp (koff = 0.024 s(-1)) to 6 bp (koff = 14 s(-1)) range. The 8 bp sequence is explored in more detail, and the NaCl dependence of kon and koff is measured. Interestingly, kon increases by >40-fold (kon = 0.10(1) s(-1) to 4.0(4) s(-1) between [NaCl] = 25 mM and 1 M), whereas in contrast, koff decreases by fourfold (0.72(3) s(-1) to 0.17(7) s(-1)) over the same range of conditions. Thus, the equilibrium constant (Keq) increases by ≈160, largely due to changes in the association rate, kon. Finally, temperature-dependent measurements reveal that increased [NaCl] reduces the overall exothermicity (ΔΔH° > 0) of duplex formation, albeit by an amount smaller than the reduction in entropic penalty (-TΔΔS° < 0). This reduced entropic cost is attributed to a cation-facilitated preordering of the two single-stranded species, which lowers the association free-energy barrier and in turn accelerates the rate of duplex formation.
In this work, the kinetics of short, fully complementary oligonucleotides are investigated at the single-molecule level. Constructs 6–9 bp in length exhibit single exponential kinetics over 2 orders of magnitude time for both forward (kon, association) and reverse (koff, dissociation) processes. Bimolecular rate constants for association are weakly sensitive to the number of basepairs in the duplex, with a 2.5-fold increase between 9 bp (k′on = 2.1(1) × 106 M−1 s−1) and 6 bp (k′on = 5.0(1) × 106 M−1 s−1) sequences. In sharp contrast, however, dissociation rate constants prove to be exponentially sensitive to sequence length, varying by nearly 600-fold over the same 9 bp (koff = 0.024 s−1) to 6 bp (koff = 14 s−1) range. The 8 bp sequence is explored in more detail, and the NaCl dependence of kon and koff is measured. Interestingly, konincreases by >40-fold (kon = 0.10(1) s−1 to 4.0(4) s−1 between [NaCl] = 25 mM and 1 M), whereas in contrast, koffdecreases by fourfold (0.72(3) s−1 to 0.17(7) s−1) over the same range of conditions. Thus, the equilibrium constant (Keq) increases by ≈160, largely due to changes in the association rate, kon. Finally, temperature-dependent measurements reveal that increased [NaCl] reduces the overall exothermicity (ΔΔH° > 0) of duplex formation, albeit by an amount smaller than the reduction in entropic penalty (−TΔΔS° < 0). This reduced entropic cost is attributed to a cation-facilitated preordering of the two single-stranded species, which lowers the association free-energy barrier and in turn accelerates the rate of duplex formation.
In this work, the kinetics of short, fully complementary oligonucleotides are investigated at the single-molecule level. Constructs 6–9 bp in length exhibit single exponential kinetics over 2 orders of magnitude time for both forward (k on , association) and reverse (k off , dissociation) processes. Bimolecular rate constants for association are weakly sensitive to the number of basepairs in the duplex, with a 2.5-fold increase between 9 bp (k′ on  = 2.1(1) × 10 6 M −1 s −1 ) and 6 bp (k′ on  = 5.0(1) × 10 6 M −1 s −1 ) sequences. In sharp contrast, however, dissociation rate constants prove to be exponentially sensitive to sequence length, varying by nearly 600-fold over the same 9 bp (k off  = 0.024 s −1 ) to 6 bp (k off  = 14 s −1 ) range. The 8 bp sequence is explored in more detail, and the NaCl dependence of k on and k off is measured. Interestingly, k on increases by >40-fold (k on  = 0.10(1) s −1 to 4.0(4) s −1 between [NaCl] = 25 mM and 1 M), whereas in contrast, k off decreases by fourfold (0.72(3) s −1 to 0.17(7) s −1 ) over the same range of conditions. Thus, the equilibrium constant (K eq ) increases by ≈160, largely due to changes in the association rate, k on . Finally, temperature-dependent measurements reveal that increased [NaCl] reduces the overall exothermicity (ΔΔH° > 0) of duplex formation, albeit by an amount smaller than the reduction in entropic penalty (−TΔΔS° < 0). This reduced entropic cost is attributed to a cation-facilitated preordering of the two single-stranded species, which lowers the association free-energy barrier and in turn accelerates the rate of duplex formation.
In this work, the kinetics of short, fully complementary oligonucleotides are investigated at the single-molecule level. Constructs 6–9 bp in length exhibit single exponential kinetics over 2 orders of magnitude time for both forward (kₒₙ, association) and reverse (kₒff, dissociation) processes. Bimolecular rate constants for association are weakly sensitive to the number of basepairs in the duplex, with a 2.5-fold increase between 9 bp (k′ₒₙ = 2.1(1) × 10⁶ M⁻¹ s⁻¹) and 6 bp (k′ₒₙ = 5.0(1) × 10⁶ M⁻¹ s⁻¹) sequences. In sharp contrast, however, dissociation rate constants prove to be exponentially sensitive to sequence length, varying by nearly 600-fold over the same 9 bp (kₒff = 0.024 s⁻¹) to 6 bp (kₒff = 14 s⁻¹) range. The 8 bp sequence is explored in more detail, and the NaCl dependence of kₒₙ and kₒff is measured. Interestingly, kₒₙincreases by >40-fold (kₒₙ = 0.10(1) s⁻¹ to 4.0(4) s⁻¹ between [NaCl] = 25 mM and 1 M), whereas in contrast, kₒffdecreases by fourfold (0.72(3) s⁻¹ to 0.17(7) s⁻¹) over the same range of conditions. Thus, the equilibrium constant (Kₑq) increases by ≈160, largely due to changes in the association rate, kₒₙ. Finally, temperature-dependent measurements reveal that increased [NaCl] reduces the overall exothermicity (ΔΔH° > 0) of duplex formation, albeit by an amount smaller than the reduction in entropic penalty (−TΔΔS° < 0). This reduced entropic cost is attributed to a cation-facilitated preordering of the two single-stranded species, which lowers the association free-energy barrier and in turn accelerates the rate of duplex formation.
In this work, the kinetics of short, fully complementary oligonucleotides are investigated at the single-molecule level. Constructs 6-9 bp in length exhibit single exponential kinetics over 2 orders of magnitude time for both forward (kon, association) and reverse (koff, dissociation) processes. Bimolecular rate constants for association are weakly sensitive to the number of basepairs in the duplex, with a 2.5-fold increase between 9 bp (k'on = 2.1(1) × 10(6) M(-1) s(-1)) and 6 bp (k'on = 5.0(1) × 10(6) M(-1) s(-1)) sequences. In sharp contrast, however, dissociation rate constants prove to be exponentially sensitive to sequence length, varying by nearly 600-fold over the same 9 bp (koff = 0.024 s(-1)) to 6 bp (koff = 14 s(-1)) range. The 8 bp sequence is explored in more detail, and the NaCl dependence of kon and koff is measured. Interestingly, kon increases by >40-fold (kon = 0.10(1) s(-1) to 4.0(4) s(-1) between [NaCl] = 25 mM and 1 M), whereas in contrast, koff decreases by fourfold (0.72(3) s(-1) to 0.17(7) s(-1)) over the same range of conditions. Thus, the equilibrium constant (Keq) increases by ≈160, largely due to changes in the association rate, kon. Finally, temperature-dependent measurements reveal that increased [NaCl] reduces the overall exothermicity (ΔΔH° > 0) of duplex formation, albeit by an amount smaller than the reduction in entropic penalty (-TΔΔS° < 0). This reduced entropic cost is attributed to a cation-facilitated preordering of the two single-stranded species, which lowers the association free-energy barrier and in turn accelerates the rate of duplex formation.
Author Holmstrom, Erik D.
Dupuis, Nicholas F.
Nesbitt, David J.
AuthorAffiliation JILA, University of Colorado and National Institute of Standards and Technology, University of Colorado, Boulder, Colorado
Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado
AuthorAffiliation_xml – name: Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado
– name: JILA, University of Colorado and National Institute of Standards and Technology, University of Colorado, Boulder, Colorado
Author_xml – sequence: 1
  givenname: Nicholas F.
  surname: Dupuis
  fullname: Dupuis, Nicholas F.
  organization: JILA, University of Colorado and National Institute of Standards and Technology, University of Colorado, Boulder, Colorado
– sequence: 2
  givenname: Erik D.
  surname: Holmstrom
  fullname: Holmstrom, Erik D.
  organization: JILA, University of Colorado and National Institute of Standards and Technology, University of Colorado, Boulder, Colorado
– sequence: 3
  givenname: David J.
  surname: Nesbitt
  fullname: Nesbitt, David J.
  email: djn@jila.colorado.edu
  organization: JILA, University of Colorado and National Institute of Standards and Technology, University of Colorado, Boulder, Colorado
BackLink https://www.ncbi.nlm.nih.gov/pubmed/23931323$$D View this record in MEDLINE/PubMed
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ContentType Journal Article
Copyright 2013 Biophysical Society
Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.
Copyright Biophysical Society Aug 6, 2013
2013 by the Biophysical Society. 2013 Biophysical Society
Copyright_xml – notice: 2013 Biophysical Society
– notice: Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.
– notice: Copyright Biophysical Society Aug 6, 2013
– notice: 2013 by the Biophysical Society. 2013 Biophysical Society
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Snippet In this work, the kinetics of short, fully complementary oligonucleotides are investigated at the single-molecule level. Constructs 6–9 bp in length exhibit...
In this work, the kinetics of short, fully complementary oligonucleotides are investigated at the single-molecule level. Constructs 6-9 bp in length exhibit...
In this work, the kinetics of short, fully complementary oligonucleotides are investigated at the single-molecule level. Constructs 6–9 bp in length exhibit...
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SubjectTerms Base Pairing
Cations - chemistry
Deoxyribonucleic acid
dissociation
Dissociative Disorders
DNA
DNA, Single-Stranded - chemistry
Kinetics
Measurement
Molecules
Oligodeoxyribonucleotides - chemistry
oligonucleotides
Proteins and Nucleic Acids
Sodium - chemistry
Sodium chloride
Temperature
Title Single-Molecule Kinetics Reveal Cation-Promoted DNA Duplex Formation Through Ordering of Single-Stranded Helices
URI https://dx.doi.org/10.1016/j.bpj.2013.05.061
https://www.ncbi.nlm.nih.gov/pubmed/23931323
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https://www.proquest.com/docview/1420163911
https://www.proquest.com/docview/1520384512
https://www.proquest.com/docview/1678557777
https://pubmed.ncbi.nlm.nih.gov/PMC3736743
Volume 105
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