Detection of enzyme activity with DNA loops

Patent metadata

Field	Value
Publication number	US-11255786-B2
Application number	US-202117348824-A
Country	US
Kind code	B2
Filing date	Jun 16, 2021
Priority date	Apr 15, 2019
Publication date	Feb 22, 2022
Grant date	Feb 22, 2022

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.

First claim

What is claimed is:

1. A method of detecting enzyme activity, the method comprising:
providing a sensor system comprising (a) a loop transducer comprising a stiffening domain of about 30 to 55 base pairs in length, a cleavage domain cleavable by an enzyme of interest, and a first hybridizing domain of about 12 to 27 base pairs in length; and (b) an output gate comprising a second hybridizing domain of about 8 to 15 base pairs in length and complementary to the first hybridizing domain, a fluorophore, and a quencher, wherein the system is configured so that in the absence of the first hybridizing domain, the quencher quenches the fluorophore, and upon hybridization of the two domains, the quencher become separated from the fluorophore sufficiently to allow fluorescence thereof;
contacting the sensor system with a sample;
allowing the sample to react with the sensor system; and then
measuring fluoresce from the fluorophore, wherein the fluorescence indicates activity of the enzyme of interest.

2. The sensor system of claim 1 , wherein the cleavage domain is cleavable by an endonuclease.

3. The sensor system of claim 1 , wherein the cleavage domain is cleavable by an protease.

4. The sensor system of claim 1 , further comprising a second output gate comprising a second fluorophore configured as a Förster resonance energy transfer partner of the other fluorophore.

Assignees

• Us Gov Sec Navy

Inventors

• Ancona Mario
• Bui Hieu

Classifications

• G01N21/6428 Primary

  Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53) · CPC title

• G01N2021/6432

  Quenching · CPC title

• G01N33/52

  Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper {and including single- and multilayer analytical elements (immunological elements G01N33/54386; involving labelled immunochemicals G01N33/58; for haemoglobin or occult blood G01N33/72)} · CPC title

• C12Q1/37 Primary

  involving peptidase or proteinase · CPC title

• G01N33/542

  with steric inhibition or signal modification, e.g. fluorescent quenching · CPC title