Enzyme transducers and sensors based on DNA loops
Inventors
Assignees
Publication Number
US-11067508-B2
Publication Date
2021-07-20
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 disclosed is a technique for enzyme detection and transduction that converts enzyme activity into a DNA signal. This DNA signal can be readily amplified, combined, and processed as information by utilizing the DNA hairpin (or stem-loop) structure and the phenomenon of strand displacement. The core structure is a loop transducer composed primarily of DNA or DNA/peptide hybrids exploiting the stiffness and topology of ultra-small circular DNAs, creating a system that responds to specific enzymes by producing a DNA oligo output.
The loop transducer comprises three functional domains: a stiffening domain of double-stranded DNA that keeps the loop open by imposing structural rigidity; a cleavage domain containing the target substrate for specific enzymes, which can be DNA, RNA, peptides, or combinations thereof; and a hybridizing domain that is initially constrained by the loop's topology and stiffness. Upon enzymatic cleavage in the cleavage domain, the loop is disrupted, releasing the hybridizing domain to interact with an output gate, such as a molecular beacon. This interaction separates a fluorophore-quencher pair, generating a detectable fluorescent signal.
The problem addressed is the need for new techniques for detecting enzyme activity with low error, high specificity, and modularity. Existing enzyme detection systems primarily provide optical or electrical outputs but often lack modularity, low error rates, or in vivo applicability. The invention solves the challenges of converting specific enzymatic activity into a DNA signal that can be polymerized, logically processed, and amplified, thereby expanding sensitivity, accuracy, and versatility of enzyme sensors.
Claims Coverage
The patent includes one independent claim defining an enzyme sensor system composed of a loop transducer and an output gate, with specific domain lengths and components. The inventive features detail structural and functional aspects enabling enzyme detection and signal transduction.
Loop transducer with specific domains and lengths
The enzyme sensor system comprises a loop transducer having a stiffening domain of about 30 to 55 base pairs in length, a cleavage domain that is cleavable by an enzyme of interest, and a first hybridizing domain of about 12 to 27 base pairs in length.
Output gate with complementary hybridizing domain and fluorescent signaling
The system includes an output gate comprising a second hybridizing domain of about 8 to 15 base pairs complementary to the first hybridizing domain, a fluorophore, and a quencher configured such that the quencher quenches the fluorophore in absence of hybridization, and upon hybridization, the quencher separates sufficiently to allow fluorescence.
The independent claim covers an enzyme sensor system that integrates a structurally defined loop transducer with a cleavage domain targeted by enzymes, and an output gate that translates hybridization events into fluorescent signals, enabling specific and sensitive enzyme detection.
Stated Advantages
Provides a general technique applicable to endonucleases, proteases, and potentially many other enzyme classes due to the non-specific mechanism based on loop stiffness and topology combined with modular design.
Allows simple, scalable design enabling processing of information beyond biomolecular sensing.
Enables straightforward coupling to DNA nanotechnology for logical processing and amplification of enzyme detection signals.
Nano-sized, non-toxic, and robust to enzymatic attack, making the system potentially suitable for in vivo applications.
Costs are low due to the ease of synthesizing oligonucleotides commercially, with expected excellent shelf-life under standard storage conditions.
The circular loop design enhances resistance to exonuclease digestion, supporting in vivo durability.
Documented Applications
Detection and monitoring of enzymes such as restriction endonucleases and proteases.
Logical processing of enzyme detection outputs using DNA logic gates (e.g., OR, NAND, AND) for improved reliability and reduction of false positives.
Amplification of DNA output signals via catalytic hairpin assembly or other enzyme-free DNA amplification methods to enhance sensitivity.
Detection of nucleic acids (ssDNA or RNA) by hybridization to the cleavage domain, enabling targeted detection of RNA biomarkers including microRNA.
Potential adaptation for detecting other biomolecules such as engineered proteins (e.g., zinc-finger nucleases).
In vivo applicability due to nano-size and robustness, facilitated by potential tethering to substrates like lipid bilayers for sensing surface-bound biomarkers.
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