Methods of Producing Competitive Aptamer FRET Reagents and Assays

A method of using a competitive type assay to determine the presence of target molecules in a solution, comprising:

• incorporating a volume of a fluorophore (“F”)-labeled aptamer into said solution that may contain unlabeled target molecules, wherein said F-labeled aptamer will bind with said target molecule, and wherein said F is located in the interior portion of said aptamer;
• adding labeled target molecules to said solution, wherein said labeled target molecules are labeled with a quencher (“Q”) that is complimentary to said F of said F-labeled aptamer, and wherein said Q-labeled target molecules compete with said unlabeled target molecules to bind with said F-labeled aptamers;
• wherein said F and said Q are spectrally matched such that there is a detectable change in the fluorescent signal of said aptamer when said F and said Q are moved into or out of functional proximity;
• wherein fluorescence light levels change proportionately in response to the amount of said Q-labeled target molecules that are able to bind with said F-labeled aptamers;
• measuring said fluorescence light level in order to determine the presence of said unlabeled target molecules in said solution;
• wherein said aptamer has a binding pocket into which said target molecule binds to said aptamer;
• and
• wherein said binding pocket is comprised of 3 to 6 nucleotides.