DNA-Encoded Libraries for Enzyme Screening via FRET
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for screening large numbers of compounds to identify those with desired biological activities are inefficient, particularly in identifying proteins with specific enzymatic activities.
Innovation Solution
A method involving nucleic acid-encoded chemical libraries, where nucleic acids with predefined sequences are expressed in vitro, exposed to reactants with fluorophores, and fluorescence energy transfer is used to detect and isolate proteins with desired enzymatic activities.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If standard screening methods are used to search for chemicals with desired biological activities, then the screening process can be performed, but the efficiency is low and the process is time-consuming
Solution Approach 1:
The patent replaces traditional mechanical/chemical screening methods with an optical detection system. Fluorophores are attached to oligonucleotides that bind to target proteins, and fluorescence resonance energy transfer (FRET) signals detect binding events automatically, eliminating manual screening steps and dramatically improving throughput while reducing time requirements
Solution Approach 2:
The patent introduces fluorophore-labeled oligonucleotides as intermediary probes. These oligonucleotides serve as mediators that bind to target proteins through sequence complementarity, and their fluorescent tags enable indirect detection of protein binding and activity, allowing efficient high-throughput screening
2Quantity of substance
If large numbers of compounds are screened to identify proteins with specific enzymatic activities, then the coverage is comprehensive, but the complexity of the screening process increases
Solution Approach 1:
The patent employs a universal oligonucleotide-probe system that can detect multiple different proteins and enzymatic activities through a single standardized platform. The fluorophore-labeled oligonucleotides serve universal binding and detection functions across diverse targets, simplifying the overall screening process despite the large number of compounds analyzed
Solution Approach 2:
The patent uses fluorescence resonance energy transfer (FRET) where fluorophores emit light at specific wavelengths upon excitation. This optical signal change provides a simple, readable output that indicates protein binding and enzymatic activity, reducing the complexity of data interpretation even when screening large compound libraries
3Measurement precision
If fluorescence detection is used to identify protein-reactant-oligonucleotide-fluorophore complexes, then the detection sensitivity is high, but the requirement for specialized equipment increases
Solution Approach 1:
The patent replaces complex biochemical assays with a simplified optical detection system. Fluorophores attached to oligonucleotides provide direct fluorescent signals upon binding to target proteins, allowing sensitive detection using standard fluorescence readers or plate readers, which are common laboratory instruments that do not require specialized equipment
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach enables the efficient screening and identification of proteins with specific enzymatic activities, facilitating the discovery of candidate molecules with desired functions.
Implementation Method 1
detecting the fluorescence of the protein-reactant-oligonucleotide-fluorophore complexes
Implementation Method 2
detecting the fluorescence energy transfer
Data Source
AI summary
Disclosed are methods, compositions and devices for screening a protein library for proteins having a desired activity, such as capable of catalyzing the formation of a bond between two reactants. In an exemplary embodiments, a plurality of proteins are expressed in vitro from a plurality of nucleic acids, the plurality of proteins are exposed with two single stranded oligonucleotides having complementary sequences, each oligonucleotide having a reactant and a fluorophore, the fluorescence of the protein-reactant-oligonucleotide-fluorophore complexes is detected and the complexes showing detectable fluorescence energy transfer are isolated, thereby isolating proteins having the desired enzymatic activity.