Detection units and methods for detecting a target analyte
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.
1. A method of detecting a target analyte in a sample, the method comprising:
(a) providing a recombinant circular DNA molecule which has been modified to hybridize to a target nucleic acid molecule, wherein one of the strands of the double-stranded DNA is discontinuous and one of the strands is continuous, wherein the discontinuous strand has a 3′ end and a 5′ end at each side of its discontinuity, wherein between 5 and 100 unpaired nucleotides at each of the 3′ and 5′ ends of the discontinuous strand do not form base pairs with the continuous strand and wherein at least some of the unpaired nucleotides in the discontinuous strand have sequence complementarity sufficient to hybridize with the target nucleic acid molecule;
(b) exposing the circular DNA to a sample containing a target analyte under conditions such that the target analyte binds to unpaired nucleotides in the discontinuous strand, wherein binding of the target analyte to the unpaired nucleotides makes the discontinuous strand become continuous and the circular DNA molecule capable of accumulating torsional stress;
(c) exposing the circular DNA molecule to a twisting agent; and
(d) detecting the supercoiling of the circular DNA molecule, wherein detection of supercoiling indicates the presence of the target analyte in the sample.
2. A method of detecting a target analyte in a sample, the method comprising:
(a) providing a circular DNA molecule having an active segment, the active segment comprising a continuous and a discontinuous strand, wherein the discontinuous strand has a 3′ end and a 5′ end, and each of the 3′ and 5′ ends has unpaired nucleotides that do not form base pairs with the continuous strand;
(b) exposing the circular DNA to a sample containing a target analyte under conditions such that the target analyte binds to the unpaired nucleotides in the discontinuous strand, wherein binding of the target analyte to the unpaired nucleotides makes the discontinuous strand become continuous and the circular DNA molecule capable of accumulating torsional stress;
(c) exposing the circular DNA to a twisting agent; and
(d) detecting the supercoiling of the circular DNA, wherein detection of supercoiling indicates the presence of the target analyte in the sample.
3. The method of claim 1 , wherein supercoiling is detected using gel electrophoresis or capillary electrophoresis.
4. The method of claim 2 , wherein supercoiling is detected using gel electrophoresis or capillary electrophoresis.
5. The method of claim 1 , wherein the twisting agent is a small molecule comprising actinomycin D, ethidium bromide, propidium, berberine, acridine or its derivatives, 9-aminoacridine, proflavine, quinacrine, daunomycin, doxorubicin, thalidomide, ellipticine, psoralen or its derivatives, the commercial dyes Gelred, Gelgreen, Sybr Gold or Sybr Green or an enzyme comprising DNA gyrase or a type II topoisomerase.
6. The method of claim 2 , wherein the twisting agent is a small molecule comprising actinomycin D, ethidium bromide, propidium, berberine, acridine or its derivatives, 9-aminoacridine, proflavine, quinacrine, daunomycin, doxorubicin, thalidomide, ellipticine, psoralen or its derivatives, the commercial dyes Gelred, Gelgreen, Sybr Gold or Sybr Green or an enzyme comprising DNA gyrase or a type II topoisomerase.
7. The method of claim 1 , wherein the target analyte is a short nucleic acid molecule comprising small interfering RNA, micro-RNA or its precursors, or fragmented DNA molecule obtained from a body fluid.
8. The method of claim 2 , wherein the target analyte is a short nucleic acid molecule comprising small interfering RNA, micro-RNA or its precursors, or fragmented DNA molecule obtained from a body fluid.
9. A method of detecting a target analyte in a sample, the method comprising:
(a) providing a circular DNA molecule with an active segment, the active segment comprising a continuous and a discontinuous strand;
(b) exposing the circular DNA to a sample containing a target analyte under conditions such that the target analyte binds to unpaired nucleotides in the active segment, and the discontinuous strand becomes continuous, and the circular DNA molecule becomes capable of accumulating torsional stress;
(c) exposing the circular DNA to a twisting agent; and
(d) detecting the supercoiling of the circular DNA, wherein detection of supercoiling indicates the presence of the target analyte in the sample.