Detection units and methods for detecting a target analyte
Inventors
Assignees
Publication Number
US-9382578-B2
Publication Date
2016-07-05
Expiration Date
2032-10-10
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Abstract
The present application relates to detection units and methods for detecting one or more target analytes in a sample. In certain embodiments, the detection unit provides a first and second surface connected by a filament which is capable of binding the target analyte in the sample. In other embodiments, the detection unit provides a circular molecule capable of binding the target analyte and accumulating torsional stress in the presence of a twisting agent. The methods provide for the detection of the target analyte through the generation of a detectable signal following the binding of the target analyte to the filament.
Core Innovation
The invention relates to detection units and methods for detecting one or more target analytes in a sample. The core concept utilizes a detection unit composed of a first and second surface connected by at least one filament, in which the filament includes an active segment capable of specifically binding a target analyte. Upon target binding, the filament undergoes a first property change, which alone may be difficult to detect due to potential non-specific interactions.
To address detection challenges, the invention introduces the application of a twisting agent or a physical rotation step after the binding event. This causes a second, more easily detectable property change, such as the accumulation of torsional stress, supercoiling, or breaking of the filament, leading to a measurable signal. For example, in embodiments using recombinant circular double-stranded DNA, target binding bridges or modifies a discontinuous strand to become continuous, thus enabling the accumulation of torsional stress which results in supercoiling upon exposure to the twisting agent.
The invention specifically addresses difficulties present in existing detection systems, such as low analyte concentrations, non-specific binding, background interference, and the inability to reliably detect short nucleic acids like micro-RNA using standard PCR. By exploiting changes in the mechanical and structural properties of the filament upon target binding and subsequent twisting or breaking, the system allows for sensitive and specific detection of target analytes, even in the presence of challenging sample backgrounds.
Claims Coverage
The patent contains two independent claims covering two main inventive features related to methods for detecting target analytes using modified circular DNA and torsional stress detection.
Detection of target analyte using modified recombinant circular DNA capable of torsional stress accumulation
A method comprising: - Providing a recombinant circular DNA molecule, wherein one strand is discontinuous and the other continuous, with between 5 and 100 unpaired nucleotides at each of the 3′ and 5′ ends of the discontinuous strand, and at least some unpaired nucleotides are complementary for hybridization with a target nucleic acid. - Exposing the circular DNA to a sample so that the target analyte binds the unpaired nucleotides, causing the discontinuous strand to become continuous and enabling the circular DNA molecule to accumulate torsional stress. - Exposing the now-continuous circular DNA to a twisting agent. - Detecting the supercoiling of the circular DNA molecule, where detection of supercoiling indicates presence of the target analyte.
Detection of target analyte using circular DNA with active segment containing continuous and discontinuous strands
A method comprising: - Providing a circular DNA molecule with an active segment made up of a continuous and a discontinuous strand, each end of the discontinuous strand having unpaired nucleotides that do not form base pairs with the continuous strand. - Exposing the DNA to a sample such that the target analyte binds the unpaired nucleotides, resulting in the discontinuous strand becoming continuous and the circular DNA capable of accumulating torsional stress. - Exposing the DNA molecule to a twisting agent. - Detecting the supercoiling of the circular DNA, where detection of supercoiling signals the presence of the target analyte.
These inventive features collectively claim methods for detecting target analytes that utilize circular DNA constructs uniquely engineered to become capable of accumulating torsional stress upon analyte binding, with the presence of the analyte detected by observing supercoiling when a twisting agent is applied.
Stated Advantages
Enables detection of very low concentrations of target analytes while reducing non-specific binding and background interference.
Allows for detection of short nucleic acids, such as micro-RNA, that are difficult to detect using PCR-based techniques.
Provides a more easily detectable signal following target binding by inducing a second property change (e.g., supercoiling) that is less likely to be caused by non-specific interactions.
Eliminates the need for PCR amplification or radioactive labeling for detection of nucleic acids.
Documented Applications
General diagnostic purposes involving detection of natural, synthetic, modified, or unmodified nucleic acids or proteins in a sample.
Detection of short nucleic acids, including micro-RNA, small interfering RNA, and fragmented DNA molecules in body fluids.
Simultaneous detection of panels of target analytes in samples by preparing multiple circular DNA molecules, each targeting a different nucleic acid.
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