Methods, systems, and compositions for studying solvent accessibility and three-dimensional structure of biological molecules
AssigneeWisconsin Alumni Research Foundation
InventorsMichael R. Sussman, J. Leon Shohet, Faraz A. Choudhury, Joshua M. Blatz, Benjamin B. Minkoff, Daniel I. Benjamin
Life of this patent
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Abstract
This disclosure provides methods, systems, and compositions of matter for studying solvent accessibility and three-dimensional structure of biological molecules. A plasma can be used to generate marker radicals, which can interact with a biological molecule and mark the solvent-accessible portions of the biological molecule.
Claims20
A method of modifying a biological molecule located in a sample, the sample contacted by a fluid or enclosed within a confined space, the sample or the fluid containing a plurality of marker radical precursors, the method comprising the following steps:
- a) generating a sequence of plasma pulses in the fluid and/or generating the sequence of plasma pulses within the sample, the sequence of plasma pulses in the fluid having at least a portion of the sequence of plasma pulses within 1 cm of the sample, thereby converting one or more of the plurality of marker radical precursors into one or more marker radicals, wherein the plasma pulses have a pulse width of between 1 ps and 1 ms, the sequence of plasma pulses have a pulse repetition rate of between 1 Hz and 100 GHz, or the sequence of plasma pulses are generated for a total length of time of between 1 ns and 1 hour, wherein the plasma of step a) is generated by a voltage in a range of between 1V and 1 MV;
- and
- b) waiting a length of time sufficient for the one or more marker radicals to interact with the biological molecule, thereby modifying the biological molecule,
- wherein steps a) and b) cause a detectable difference in the biological molecule without initiating denaturation of the biological molecule.
A method of determining if a portion of a biological molecule is accessible to a solvent, the biological molecule and the solvent contained in a sample, the sample contacted by a fluid or enclosed within a confined space, the sample or the fluid containing a marker radical precursor, the method comprising the following steps:
- a) oxidizing, by way of a plasma that introduces marker radicals to the sample, the biological molecule, wherein the oxidizing occurs in the presence of oxygen, wherein the oxidizing occurs while the biological molecule is in solution within the solvent, and wherein the plasma is configured to elevate a temperature of the sample by an amount less than would begin denaturation of the biological molecule;
- b) subsequent to step a), assessing whether the portion of the biological molecule was oxidized by the oxidizing of step a), wherein the presence of oxidizing indicates that the portion is accessible to the solvent and the absence of oxidizing indicates that the portion is inaccessible to the solvent;
- and
- c) generating a report indicating whether the portion is accessible to the solvent or inaccessible to the solvent.
A method of assessing a biological sample containing one or more biological molecules having one or more solvent accessible portions and one or more solvent inaccessible portions, the method comprising the following steps:
- a) acquiring a first subsample and a second subsample of the biological sample, the first sub sample and the second subsample containing substantially equivalent concentrations of the one or more biological molecules;
- b) introducing a denaturing factor into the second subsample of the biological sample, the denaturing factor configured to alter at least a portion of the one or more biological molecules located within the second subsample to expose at least a portion of the solvent inaccessible portions to solvent;
- c) oxidizing, by way of a plasma that introduces marker radicals to the first subsample and the second subsample, the one or more biological molecules in the first subsample and the second subsample, wherein the plasma is configured to elevate a temperature of the first sub sample and the second subsample by an amount less than would begin denaturation of the one or more biological molecules;
- d) subsequent to step c), assessing a difference in oxidization levels between the one or more biological molecules in the first subsample and the second subsample, thereby identifying at least a portion of the one or more solvent inaccessible portions;
- and
- e) generating a report indicating the identification of the at least a portion of the one or more solvent inaccessible portions.
Dependencies are inferred from the claim text.