Optimization of ligand affinity for RNA targets using mass spectrometry
AssigneeIsis Pharmaceuticals, Inc.
InventorsRichard Griffey, Steven Hofstadler, Jared J. Drader, Kristin S. Lowery
Life of this patent
Estimating the term…
Abstract
The present invention provides methods for the identification ligand compounds that bind to target molecules such as proteins or structured RNA with as little as millimolar (mM) affinity using mass spectrometry. The methods may be used to determine the mode of binding interaction between two or more of these ligand compounds to the target as well as their relative affinities. Also provided are methods for designing compounds having greater affinity to a target molecule by identifying two or more ligands using mass spectrometry methods of the invention and linking the ligands together to form a novel compound.
Claims26
A method for identifying at least one ligand having an affinity for an RNA target molecule that is equal to or greater than a baseline affinity comprising:
- mixing an amount of ammonium with an excess amount of the RNA target molecule such that unbound RNA target molecule is present in the mixture;
- introducing the mixture of ammonium and RNA target molecule into a mass spectrometer;
- adjusting the mass spectrometer desolvation conditions such that the signal strength of the ammonium bound to the RNA target molecule is from 1% to about 30% of the signal strength of unbound RNA target molecule;
- introducing at least one ligand into a test mixture of the RNA target molecule and the ammonium;
- introducing the test mixture into the mass spectrometer;
- and
- identifying any complexes of the ligand and the RNA target wherein the ligand has greater than the baseline affinity for the RNA target molecule by discerning those signals that have a signal strength greater than the background noise of the mass spectrometer.
A method for identifying at least one ligand having an affinity for an RNA that is equal to or greater than a baseline affinity comprising:
- selecting an ammonium ion that forms a non-covalent binding complex with said RNA;
- mixing an amount of said ammonium ion with an excess amount of said RNA such that unbound RNA is present in said mixture;
- introducing said mixture of said ammonium ion and said RNA into an electrospray mass spectrometer, wherein said electrospray mass spectrometer includes a desolvation capillary and a lens element;
- adjusting the electrospray mass spectrometer desolvation conditions such that the signal strength of said ammonium ion bound to said RNA is from 1% to about 30% of the signal strength of unbound RNA, wherein said adjusting desolvation conditions comprises adjustment of the voltage potential across said capillary and said lens element generates an abundance of an ion from a monoammonium-RNA complex to from about 10% to about 20% of the abundance of the ion from target RNA;
- introducing at least one ligand into a test mixture of said RNA and said ammonium ion;
- introducing said test mixture into said electrospray mass spectrometer;
- and
- identifying any complexes of said ligand and said RNA wherein said ligand has greater than said baseline affinity for said RNA by discerning those signals that have a signal strength greater than the background noise of the electrospray mass spectrometer.
Dependencies are inferred from the claim text.