Detection of enzyme activity with DNA loops
Inventors
Assignees
Publication Number
US-11255786-B2
Publication Date
2022-02-22
Expiration Date
2040-04-14
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Abstract
The stiffness and topology of ultra-small circular DNAs and DNA/peptide hybrids are exploited to create a transducer of enzyme activity with low error rates. The modularity and flexibility of the concept are illustrated by demonstrating various transducers that respond to either specific restriction endonucleases or to specific proteases. In all cases the output is a DNA oligo signal that, as we show, can readily be converted directly to an optical readout, or can serve as input for further processing, for example, using DNA logic or amplification. By exploiting the DNA hairpin (or stem-loop) structure and the phenomenon of strand displacement, an enzyme signal is converted into a DNA signal, in the manner of a transducer. This is valuable because a DNA signal can be readily amplified, combined, and processed as information.
Core Innovation
The invention describes a technique for detecting enzyme activity by converting an enzyme signal into a DNA signal. This conversion exploits the stiffness and topology of ultra-small circular DNAs and DNA/peptide hybrids, using a loop transducer consisting of three functional domains: a stiffening domain formed by a double-stranded DNA segment to maintain loop rigidity, a cleavage domain recognized and cleaved by the enzyme of interest, and a hybridizing domain that interacts with an output gate such as a molecular beacon. The intact loop constrains the hybridizing domain due to mechanical and topological stress, preventing premature signaling.
Upon cleavage by the target enzyme in the cleavage domain, the loop opens, relieving stress and allowing the hybridizing domain to bind to the output gate via strand displacement. This interaction separates a fluorophore from a quencher within the output gate, generating a fluorescent signal proportional to enzyme activity. The DNA signal output can be further processed, combined with other signals using DNA logic operations, or amplified using DNA-based amplification techniques, enabling sensitive and modular enzyme detection.
The problem addressed by this invention stems from the need for improved enzyme detection methods. Existing enzyme detection systems often rely on optical or electrical readouts that may have limitations in specificity, sensitivity, cost, or in vivo applicability. The invention meets the need for new, general techniques capable of detecting various enzymes including endonucleases and proteases with low error rates. By converting enzyme activity into a DNA output signal, the method leverages the well-established DNA nanotechnology toolbox for downstream amplification and logical processing, thereby enhancing detection capability and modularity.
Claims Coverage
The patent presents one independent claim defining a method of detecting enzyme activity using a sensor system composed of a loop transducer and an output gate, incorporating specific structural and functional features.
Loop transducer configuration
The sensor system includes a loop transducer comprising a stiffening domain about 30 to 55 base pairs in length, a cleavage domain cleavable by an enzyme of interest, and a first hybridizing domain about 12 to 27 base pairs in length.
Output gate design
An output gate comprises a second hybridizing domain about 8 to 15 base pairs in length complementary to the first hybridizing domain, a fluorophore, and a quencher, configured so that in the absence of hybridization fluorescence is quenched, and upon hybridization the fluorophore and quencher are separated enabling fluorescence.
Method steps for detecting enzyme activity
The method involves providing the sensor system, contacting it with a sample, allowing reaction, and measuring fluorescence from the fluorophore, where fluorescence indicates enzyme activity.
The claims focus on the construction and operation of a loop transducer with specified domain lengths and a complementary output gate that transduces enzymatic cleavage into a measurable fluorescence signal, enabling detection of enzyme activity.
Stated Advantages
The method provides a general, modular technique applicable to different classes of enzymes such as endonucleases and proteases, enabled by the non-specific mechanism based on loop stiffness and topology.
Its simplicity makes the design scalable and suitable for processing biological information and other applications with DNA-based logic and amplification.
The DNA oligo output can be integrated easily with existing DNA nanotechnology methods including strand displacement networks for logic processing and signal amplification.
The nano-sized, non-toxic, and enzyme-robust nature of the system makes it adaptable for in vivo applications unlike many existing detection methods.
The approach offers low-cost implementation due to the commercial availability of synthetic oligomers and exhibits excellent shelf-life under standard storage.
The circular DNA loop design provides resistance to exonuclease digestion, enhancing suitability for in vivo use.
Documented Applications
Detection and monitoring of enzymes such as proteases and restriction endonucleases, important for biological and medical diagnostic applications.
Combination of outputs from multiple loop transducers to perform DNA logic operations like OR, NAND, and AND gates for improved reliability and complex sensing.
Amplification of enzyme detection signals at the molecular level using DNA-based enzyme-free amplification schemes such as catalytic hairpin assembly.
Detection of nucleic acids such as ssDNA or RNA by making loop transducers susceptible to cleavage upon hybridization with the target nucleic acid.
Potential detection of microRNAs, engineered proteins such as zinc-finger nucleases, and extension to various biomedical and homeland security sensing applications.
Interested in licensing this patent?