Protease transducers and sensors based on DNA loops
Inventors
Assignees
Publication Number
US-11150186-B1
Publication Date
2021-10-19
Expiration Date
2040-04-14
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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 an enzyme sensor system that utilizes the stiffness and topology of ultra-small circular DNAs and DNA/peptide hybrids to create a transducer of enzyme activity with low error rates. This transducer converts enzyme signals into DNA signals using DNA hairpin (stem-loop) structures and strand displacement. This conversion enables the DNA signals to be readily amplified, combined, or processed for further use such as generating fluorescence output or DNA logic operations.
The system consists of a loop transducer comprising three distinct functional domains: a stiffening domain formed by a double-stranded DNA segment creating tension and topological constraint on the loop, a cleavage domain containing the target substrate of the enzyme (which can be DNA, RNA, peptide, or a hybrid), and a hybridizing domain of single-stranded DNA complementary to the output gate. Enzymatic cleavage of the loop relieves the loop stress and topological constraint, releasing the hybridizing domain that then opens the output gate hairpin via strand displacement, resulting in a fluorescence signal.
The problem being solved is the need for new techniques to detect and monitor enzyme activity with high specificity and low error rates, which is valuable in biology and medicine due to enzymes' essential roles in health and disease. Existing enzyme detection systems generally rely on optical or electrical outputs but may lack modularity, in vivo applicability, or signal processing capability. The present invention addresses this by providing a general, modular, and scalable method to transduce enzyme activity into a robust DNA signal that integrates well with DNA nanotechnology for amplification and logic processing.
Claims Coverage
The patent includes one independent claim that defines the core components and configuration of an enzyme sensor system.
Loop transducer with specific domain structure
The sensor system comprises a loop transducer having a stiffening domain ranging from about 30 to 55 base pairs, a cleavage domain cleavable by a protease, and a first hybridizing domain of about 12 to 27 base pairs in length.
Output gate with fluorescence signaling
The output gate includes a second hybridizing domain complementary to the first hybridizing domain, a first fluorophore, and a quencher arranged so that the fluorophore is quenched in the absence of hybridization but fluoresces upon hybridization-induced separation from the quencher.
The inventive features collectively disclose a protease sensor system that operates via a DNA loop transducer combined with a fluorescence-based output gate, enabling sensitive and specific enzymatic activity detection through DNA hybridization and strand displacement mechanisms.
Stated Advantages
The method provides a general technique applicable to endonucleases, proteases, and potentially many other enzyme classes, demonstrating modularity and broad applicability.
The loop transducer design is simple and scalable, allowing the processing of information beyond biomaterials.
The DNA oligo output readily couples with DNA nanotechnology, facilitating logical processing and amplification of sensing outputs.
The nano-sized, non-toxic, and enzymatically robust nature of the system makes it adaptable for in vivo applications.
The approach is low cost due to the ease of commercial oligomer synthesis and exhibits excellent shelf-life under standard storage conditions.
The circular loop design confers resistance to exonuclease digestion, enhancing in vivo stability.
Documented Applications
Detection and monitoring of enzyme activity such as specific restriction endonucleases (e.g., HaeIII, NcoI-HF) and proteases (e.g., trypsin, tobacco etch virus protease).
Implementation of logical operations on enzyme activity signals, including Boolean OR, NAND, and AND gates, by combining outputs of multiple loop transducers.
Amplification of DNA output signals via catalytic hairpin assembly (CHA), enabling sensitive detection at low concentrations.
Detection of nucleic acids such as ssDNA or RNA biomarkers by hybridization to the cleavage domain to trigger endonuclease cleavage and signal generation.
Potential future extensions include use with microRNAs, engineered proteins (e.g., zinc-finger nucleases), and alternate readout modalities such as color-change or electrical outputs.
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