Thermodynamic and kinetic characterization of antisense oligodeoxynucleotide binding to a structured mRNA

S. Patrick Walton, Gregory N. Stephanopoulos, Martin L. Yarmush, Charles M. Roth

Research output: Contribution to journalArticle

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Abstract

Antisense oligonucleotides act as exogenous inhibitors of gene expression by binding to a complementary sequence on the target mRNA, preventing translation into protein. Antisense technology is being applied successfully as a research tool and as a molecular therapeutic. However, a quantitative understanding of binding energetics between short oligonucleotides and longer mRNA targets is lacking, and selecting a high-affinity antisense oligonucleotide sequence from the many possibilities complementary to a particular RNA is a critical step in designing an effective antisense inhibitor. Here, we report measurements of the thermodynamics and kinetics of hybridization for a number of oligodeoxynucleotides (ODNs) complementary to the rabbit β-globin (RBG) mRNA using a binding assay that facilitates rapid separation of bound from free species in solution. A wide range of equilibrium dissociation constants were observed, and association rate constants within the measurable range correlated strongly with binding affinity. In addition, a significant correlation was observed of measured binding affinities with binding affinity values predicted using a thermodynamic model involving DNA and RNA unfolding, ODN hybridization, and RNA restructuring to a final free energy minimum. In contrast to the behavior observed for hybridization of short strands, the association rate constant increased with temperature, suggesting that the kinetics of association are related to disrupting the native structure of the target RNA. The rate of cleavage of the RBG mRNA in the presence of ribonuclease H and ODNs of varying association kinetics displayed apparent first-order kinetics, with the rate constant exhibiting binding-limited behavior at low association rates and reaction-limited behavior at higher rates. Implications for the rational design of effective antisense reagents are discussed.

Original languageEnglish (US)
Pages (from-to)366-377
Number of pages12
JournalBiophysical Journal
Volume82
Issue number1
StatePublished - 2002
Externally publishedYes

Profile

Oligodeoxyribonucleotides
Thermodynamics
Messenger RNA
RNA
beta-Globins
Antisense Oligonucleotides
Rabbits
RNA Folding
Ribonuclease H
Protein Biosynthesis
Oligonucleotides
Technology
Gene Expression
Temperature
DNA
Proteins

ASJC Scopus subject areas

  • Biophysics

Cite this

Thermodynamic and kinetic characterization of antisense oligodeoxynucleotide binding to a structured mRNA. / Patrick Walton, S.; Stephanopoulos, Gregory N.; Yarmush, Martin L.; Roth, Charles M.

In: Biophysical Journal, Vol. 82, No. 1, 2002, p. 366-377.

Research output: Contribution to journalArticle

Patrick Walton, S.; Stephanopoulos, Gregory N.; Yarmush, Martin L.; Roth, Charles M. / Thermodynamic and kinetic characterization of antisense oligodeoxynucleotide binding to a structured mRNA.

In: Biophysical Journal, Vol. 82, No. 1, 2002, p. 366-377.

Research output: Contribution to journalArticle

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