From Rutgers: “Efficient and Cost-effective Method for Quantifying Nucleic Acid Binding Strength”

Rutgers University
Rutgers University

For Information, Contact:
Shan Wan
Rutgers the State University of New Jersey
Kenneth Breslauer
G. Eric Plum
Craig Gelfand

Invention Summary
Rutgers University investigators, led by Dr. Kenneth Breslauer, have developed a novel method for quantifying the stability of nucleic acid duplexes, based on competitive equilibrium measurements. The method, depicted at right, employs a fluorescently labeled duplex which provides both the observable signal and a common reference for the experiments. Through competitive equilibria, the nucleic acid being evaluated (X), which is wholly or partially complementary to one strand of the reference duplex, displaces one strand of the reference duplex, altering the fluorescence signal. The X concentration dependence of the change in fluorescence is readily evaluated to yield the relative thermodynamic stabilities of the two duplexes at high precision. If the X concentration is unknown, the method can be adapted to measure it.

The method is ‘label-free’ in that the nucleic acid being screened (X) requires no modification, yielding a cost-effective screening method. The method is limited only by the lower limit of detection of the fluorescence observable, thus screening of unknowns for binding efficiency consumes very little material. The method provides higher precision binding affinity measurements than conventional spectroscopic or calorimetric methods, while requiring only a fraction of the material. Being based on simple fluorescence measurements, the method is readily adapted to high-throughput screening approaches.

The method is intrinsically flexible regarding the molecules to be evaluated. For example, binding can be tested for virtually any non-Watson-Crick base-pairing, including, but not limited to, single or multiple nucleotide mismatches, damaged or synthetically modified nucleotides, DNA or RNA folding motifs, polymorphisms, and even entirely non-natural nucleic acid mimetics.
Market Application
• Nucleic acid-targeted therapies: The nucleic acids being screened can be natural or synthetic (e.g. mimetics) intended for therapeutic benefit (e.g. siRNA, promoter site blockers, etc.) The method could have utility during initial screening or during target molecule refinement/optimization.

• In vitro diagnostics

o Screening for single nucleotide polymorphisms (SNPs), or single-base or longer DNA mutations

o DNA or RNA quantification, e.g. viral load

o DNA identification, e.g. bacterial strain identification, donor tissue matching, etc.

o Due to the ability to screen at low concentrations, prior amplification may not be required.
• Simplicity: stability, concentration, and mutation data all obtainable from a single experiment

• High sensitivity

• Economical: less material required, and label-free with respect to the ‘unknowns’ being assessed.

• Time-saving and easily adapted to high-throughput screening.
Intellectual Property & Development Status
Patents issued: US Patents 6815163 and 7468250. Method fully described in Proc Natl Acad Sci U S A. 1999 May 25;96(11):6113-8. Available for licensing and/or research collaboration.

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