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 in | Biophysical journal Vol. 105; no. 3; pp. 756 - 766 |
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Main Authors | , , |
Format | Journal Article |
Language | English |
Published |
United States
Elsevier Inc
06.08.2013
Biophysical Society The Biophysical Society |
Subjects | |
Online Access | Get full text |
ISSN | 0006-3495 1542-0086 1542-0086 |
DOI | 10.1016/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. |
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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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DOI | 10.1016/j.bpj.2013.05.061 |
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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 https://www.proquest.com/docview/1421972556 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 |
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