Molecular biosensors capable of signal amplification
Inventors
Chang, Yie-Hwa • Tian, Ling • Heyduk, Tomasz
Assignees
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Publication Number
US-9797892-B2
Publication Date
2017-10-24
Expiration Date
Abstract
The present invention provides molecular biosensors capable of signal amplification, and methods of using the molecular biosensors to detect the presence of a target molecule.
Core Innovation
The disclosed invention is a molecular biosensor made of a plurality of constructs including antibody or antibody fragment binding regions for two distinct epitopes on a polypeptide or protein and corresponding flexible linkers. The antibody binding to the polypeptide or protein brings nucleotide sequences into association such that, in the absence of the polypeptide or protein, R7 and the oligonucleotide construct do not produce detectable signal.
When the polypeptide or protein is present, complementary nucleotide sequences associate to create a restriction endonuclease recognition site. Upon recognition and cleavage of the restriction endonuclease site, the signaling molecule R7 dissociates to produce detectable signal.
The disclosure further includes a restriction endonuclease-based molecular biosensor architecture in which the restriction enzyme is part of the system with a plurality of constructs, and antibody binding enables formation of the restriction endonuclease site. The biosensor includes a solid support, and the signaling molecule is configured to dissociate upon cleavage to generate the detectable signal.
Claims Coverage
The document includes two independent claims. Across the independent claims, the inventive features are: antibody/antibody-fragment target-dependent restriction endonuclease site formation using complementary oligonucleotide regions, cleavage-triggered dissociation of a signaling molecule, and configuration that yields no detectable signal in the absence of the target; the second independent claim also includes the restriction enzyme as part of the biosensor system.
Target-dependent restriction endonuclease site formation with complementary oligonucleotide regions
The biosensor includes antibody or antibody fragment that binds to a first epitope and antibody or antibody fragment that binds to a second epitope, and nucleotide sequences that associate with an oligonucleotide construct when the polypeptide or protein is present, thereby creating a restriction endonuclease site that is cleaved by a restriction endonuclease that recognizes the restriction endonuclease site.
Cleavage-triggered dissociation of a signaling molecule to produce detectable signal
In the presence of the polypeptide or protein, nucleotide sequences associate with the oligonucleotide construct to create a restriction endonuclease site; upon cleavage of that site, the signaling molecule dissociates, producing detectable signal, while in the absence of the polypeptide or protein the signaling molecule and oligonucleotide construct do not produce detectable signal.
Signal generation configured with signaling molecule and solid support release
The biosensor includes a signaling molecule and a solid support such that cleavage of the restriction endonuclease site causes dissociation of the signaling molecule to generate detectable signal.
Restriction enzyme included in the molecular biosensor system for detection
A molecular biosensor includes a restriction enzyme and a plurality of constructs, where antibody binding to two epitopes enables formation of a restriction endonuclease site comprising complementary regions, and upon cleavage the signaling molecule dissociates to produce detectable signal.
Both independent claims cover the same core architecture: antibody or antibody fragment binding to two distinct epitopes enables target-dependent assembly of a restriction endonuclease site using complementary nucleotide regions; cleavage of that site triggers dissociation of a signaling molecule to generate detectable signal with no signal in the absence of the target. The second independent claim additionally frames the system as including the restriction enzyme.
Stated Advantages
Produces detectable signal in the presence of a target polypeptide or protein.
Does not produce detectable signal in the absence of the polypeptide or protein.
Generates signal via dissociation of the signaling molecule upon cleavage of a restriction endonuclease site.
Documented Applications
Determining whether a polypeptide or protein is present in a sample by combining the sample with the molecular biosensor, contacting with a restriction endonuclease that recognizes a double-stranded recognition site, and measuring release of the signaling molecule.
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