Recent technological advances in the biomedical sciences have made it possible to take inventory of the components of biological systems at an unprecedented level of detail. This applies not only to genes and genetic regulatory elements, but also to the proteins and small molecules that exist within living organisms. Critical to understanding the functional interplay of these components, in both health and disease, is the quantitative measurement of their concentration in biological samples. For genes and their transcription products this is readily accomplished through techniques such as the quantitative polymerase chain reaction (qPCR), which have revolutionized molecular biology and clinical diagnostics. For proteins and small molecules, however, the analytical methods are more specialized, and do not benefit from exponential signal amplification as occurs with qPCR. During the previous project period, a new approach was developed for the quantitative detection of proteins and small molecules that has high specificity and achieves high sensitivity due to exponential signal amplification. The approach relies on a novel class of self-replicating nucleic acid enzymes that undergo exponential amplification at constant temperature, and can be made to do so contingent upon recognition of a target ligand. The proposed research will expand the generality of the system so that it can be applied to a broad range of targets, including disease-related proteins, drugs, and metabolites. A real-time fluorescent assay will be developed that enables high-throughput, multiplex analysis using standard instrumentation. The self-replicating enzymes will be optimized using a combination of site-specific chemical modification and directed evolution so that they are stable in biological samples and operate with high catalytic efficiency. The method for linking the ligand-recognition element to the replicating enzymes will be generalized and applied to a variety of proteins of relevance to biosensing and clinical diagnostics. The proposed research also will investigate a potentially more far-reaching approach that takes advantage of the unique ability of the replicating enzymes to evolve in a self-sustained manner, enabling them to configure themselves to recognize a target molecule. This is analogous to the maturation of antigen recognition by the immune system, but would occur entirely in the test tube within the context of a synthetic genetic system. These efforts will provide the opportunity to develop a new class of smart materials to help keep pace with the rapid rate of discovery of novel biological targets.

Public Health Relevance

This proposal concerns a new and general approach for measuring the concentrations of proteins and small molecules in biological samples. It relies on a novel class of self-replicating nucleic acid enzymes that undergo exponential amplification at constant temperature, contingent upon recognition of a target molecule. This approach could have broad application in biosensing and clinical diagnostics, enabling one to measure disease-related proteins, drugs, and metabolites with high sensitivity and specificity.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM065130-10
Application #
8387774
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Gerratana, Barbara
Project Start
2002-04-01
Project End
2015-11-30
Budget Start
2012-12-01
Budget End
2013-11-30
Support Year
10
Fiscal Year
2013
Total Cost
$347,448
Indirect Cost
$164,098
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037
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Robertson, Michael P; Joyce, Gerald F (2014) Highly efficient self-replicating RNA enzymes. Chem Biol 21:238-45
Breaker, Ronald R; Joyce, Gerald F (2014) The expanding view of RNA and DNA function. Chem Biol 21:1059-65
Ferretti, Antonio C; Joyce, Gerald F (2013) Kinetic properties of an RNA enzyme that undergoes self-sustained exponential amplification. Biochemistry 52:1227-35
Sczepanski, Jonathan T; Joyce, Gerald F (2013) Binding of a structured D-RNA molecule by an L-RNA aptamer. J Am Chem Soc 135:13290-3
Olea Jr, Charles; Horning, David P; Joyce, Gerald F (2012) Ligand-dependent exponential amplification of a self-replicating L-RNA enzyme. J Am Chem Soc 134:8050-3
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