Methods of using molecular constructs for detection of biochemical reactions
View Patent ↗The present invention relates to molecular constructs and methods of their use in detecting biochemical reactions. In particular, the invention relates to a molecular construct having a capture portion and a substrate portion, where the capture portion isolates the construct from a sample medium, and the substrate portion enables the construct to be acted upon and undergo a physical change which can be detected and measured. These molecular constructs may be used in diagnostic assays, genetic screening for potential risks of certain diseases in individuals, and drug discovery and toxicogenomics, using high throughput screening of compounds.
1. A method of detecting a plurality of enzyme activities in a sample, comprising:
(a) adding a plurality of types of PNA-substrate chimerae to the sample, said PNA-substrate chimerae having a PNA capture portion and a substrate for an enzyme, wherein different types have different substrates capable of reacting with different enzymes but each type has a particular substrate and a selected PNA sequence capture portion;
(b) allowing the enzymes to react with the substrate portions of the chimera population;
(c) capturing the reacted PNA-substrate chimerae with different types of oligonucleotides, wherein said different types of oligonucleotides are displayed on differently encoded microparticles, and wherein said capturing is by annealing of a type of oligonucleotide to a selected PNA sequence capture portion; and
(d) detecting the changes in the substrates of captured reacted PNA-substrate chimerae;
(e) decoding the encoded microparticles to identify which substrates reacted and thereby identify the presence of particular enzymes in the sample.
2. The method of claim 1 , wherein the microparticles are color-encoded.
3. The method of claim 1 , wherein the PNA-substrate chimera contains two tags.
4. The method of claim 3 , wherein the tags are a fluorescent moiety.
5. The method of claim 1 , wherein the substrate portion of the PNA-substrate chimera is a substrate of a caspase.
6. The method of claim 1 , wherein the substrate portion of the PNA-substrate chimera is a substrate of a phosphatase.
7. The method of claim 1 , wherein the capture portion of the PNA-substrate chimera is a bis-PNA.