LABEL-FREE FUNCTIONAL NUCLEIC ACID SENSORS FOR DETECTING TARGET AGENTS
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IPC분류정보
국가/구분
United States(US) Patent
공개
국제특허분류(IPC7판)
G01N-021/77
C07H-021/02
C07H-021/04
출원번호
US-0267414
(2011-10-06)
공개번호
US-0252128
(2012-10-04)
발명자
/ 주소
Lu, Yi
Xiang, Yu
Xu, Weichen
출원인 / 주소
The Board of Trustees of the University of Illinois
인용정보
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초록▼
A general methodology to design label-free fluorescent functional nucleic acid sensors using a vacant site approach and an abasic site approach is described. In one example, a method for designing label-free fluorescent functional nucleic acid sensors (e.g., those that include a DNAzyme, aptamer or
A general methodology to design label-free fluorescent functional nucleic acid sensors using a vacant site approach and an abasic site approach is described. In one example, a method for designing label-free fluorescent functional nucleic acid sensors (e.g., those that include a DNAzyme, aptamer or aptazyme) that have a tunable dynamic range through the introduction of an abasic site (e.g., dSpacer) or a vacant site into the functional nucleic acids. Also provided is a general method for designing label-free fluorescent aptamer sensors based on the regulation of malachite green (MG) fluorescence. A general method for designing label-free fluorescent catalytic and molecular beacons (CAMBs) is also provided. The methods demonstrated here can be used to design many other label-free fluorescent sensors to detect a wide range of analytes. Sensors and methods of using the disclosed sensors are also provided.
대표청구항▼
1. A sensor, comprising a catalytic nucleic acid molecule specific for a target agent comprising an enzyme nucleic acid strand and a substrate nucleic acid strand,wherein the enzyme nucleic acid strand comprises a 3′-end and a 5′-end and an active site specific for a target agent,wherein the substra
1. A sensor, comprising a catalytic nucleic acid molecule specific for a target agent comprising an enzyme nucleic acid strand and a substrate nucleic acid strand,wherein the enzyme nucleic acid strand comprises a 3′-end and a 5′-end and an active site specific for a target agent,wherein the substrate nucleic acid strand comprises a 3′-end and a 5′-end,wherein the substrate nucleic acid strand comprises nucleotides at the 5′-end of the substrate nucleic acid strand that permits formation of a loop at the 5′-end of the substrate nucleic acid strand, andwherein the substrate nucleic acid strand hybridizes with the enzyme nucleic acid strand, thereby forming a vacant site between the 3′-end of the enzyme nucleic acid strand and the 5′-end of the substrate nucleic acid strand, wherein the vacant site is opposite to a cytosine present in the substrate nucleic acid strand. 2. The sensor of claim 1, wherein the vacant site is flanked by guanines present on a 3′-nucleotide of the enzyme nucleic acid strand and a 5′-nucleotide of the substrate nucleic acid strand. 3. The sensor of claim 1, wherein the loop is formed by at least 6 nucleotides between the vacant site and the cytosine opposite to the vacant site. 4. The sensor of claim 1, wherein at least one pair of nucleotides are mismatched upon hybridization of the substrate nucleic acid molecule with the enzyme nucleic acid strand. 5. The sensor of claim 1, wherein in the absence of the target agent, fluorescence of a fluorophore bound to the vacant site is quenched, and wherein in the presence of the target agent, catalytic cleavage of substrate nucleic acid strand perturbs the vacant site and release the fluorophore bound to the vacant site resulting in increased fluorescence. 6. A sensor, comprising an aptamer specific for a target agent comprising a 3′-end and a 5′-end;a first nucleic acid molecule having a 3′-end and a 5′-end, wherein the 5′-end of the aptamer is attached to the 3′-end of the first nucleic acid molecule, wherein the 3′-end of the first nucleic acid comprises the nucleotide sequence NCN, wherein N is any nucleotide, and wherein the 5′-end of the first nucleic acid molecule forms a loop; anda second nucleic acid molecule having a 3′-end and a 5′-end, wherein the second nucleic acid molecule is hybridized to the 5′-end of the aptamer, thereby forming a vacant site between the 5′-end of the first nucleic acid molecule and the 3′-end of the second nucleic acid molecule, wherein the vacant site is opposite to the cytosine of the NCN sequence of the first nucleic acid molecule. 7. The sensor of claim 6, wherein the vacant site is flanked by guanines present on a 3′-nucleotide of the second nucleic acid molecule and a 5′-nucleotide of the first nucleic acid molecule. 8. The sensor of claim 6, wherein the loop is formed by at least 6 nucleotides between the vacant site and the cytosine opposite to the vacant site. 9. The sensor of claim 6, wherein in the absence of the target agent, fluorescence of a fluorophore bound to the vacant site is quenched, and wherein in the presence of the target agent, a conformational change in the aptamer perturbs the vacant site and release the fluorophore bound to the vacant site resulting in increased fluorescence. 10. A sensor, comprising a catalytic nucleic acid specific for a target agent comprising an enzyme nucleic acid strand and a substrate nucleic acid strand,wherein the enzyme nucleic acid strand comprises a 5′-end and a 3′-end and an active site specific for a target agent,wherein the substrate nucleic acid strand comprises a 3′-end and a 5′-end and comprises a cytosine 2 to 6 nucleotides from the 5′-end of the substrate nucleic acid strand,wherein a nucleotide 2 to 6 nucleotides from the 3′-end of the enzyme nucleic acid strand is replaced by an abasic site, andwherein the substrate nucleic acid strand hybridizes with the enzyme nucleic acid strand, resulting in the abasic site being opposite to the cytosine 2 to 6 nucleotides from the 5′-end of the substrate nucleic acid strand. 11. The sensor of claim 10, wherein the abasic site is flanked by guanines. 12. The sensor of claim 10, wherein at least one pair of nucleotides are mismatched upon hybridization of the substrate nucleic acid strand with the enzyme nucleic acid strand. 13. The sensor of claim 10, wherein in the absence of the target agent, fluorescence of a fluorophore bound to the abasic site is quenched, and wherein in the presence of the target agent, catalytic cleavage of substrate nucleic acid strand perturbs the abasic site and releases the fluorophore bound to the abasic site resulting in increased fluorescence. 14. A sensor, comprising: an aptamer specific for a target agent comprising a 3′-end and a 5′-end;a first nucleic acid molecule comprising at least four nucleotides attached to the 3′-end of the aptamer; anda second nucleic acid molecule comprising a 5′-end and a 3′-end and an abasic site, wherein second nucleic acid molecule has complementarity to the aptamer and to the first nucleic acid molecule, wherein the second nucleic acid molecule is hybridized to the aptamer and the first nucleic acid molecule the abasic site is opposite to a cytosine in the aptamer or first nucleic acid molecule. 15. The sensor of claim 14, wherein in the absence of the target agent, fluorescence of a fluorophore bound to the abasic site is quenched, and wherein in the presence of the target agent, a conformational change in the aptamer perturbs the abasic site and release the fluorophore bound to the abasic site resulting in increased fluorescence. 16. A sensor, comprising: an aptamer specific for a target agent;a malachite green (MG) RNA aptamer, wherein the malachite green RNA aptamer is linked to the aptamer specific for the target agent; anda bridging nucleic acid strand, wherein the bridging strand is complementary to consecutive nucleotides present in the aptamer specific for the target and the malachite green RNA aptamer, and upon hybridization of the bridging nucleic acid strand to the aptamer specific for the target and the malachite green RNA aptamer forms a stable complex to prevent the malachite green aptamer strand from binding malachite green if there is no target agent is present. 17. The sensor of claim 16, wherein in the absence of the target agent, fluorescence from MG is decreased due to an inability of MG to bind to MG RNA aptamer, and wherein in the presence of the target agent, a conformational change in the aptamer specific for the target releases the bridging strand resulting in increased MG fluorescence. 18. A sensor, comprising a catalytic nucleic acid specific for a target agent comprising an enzyme nucleic acid strand and a substrate nucleic acid strand,wherein the enzyme nucleic acid strand comprises a 5′-end and a 3′-end and an active site specific for a target agent,wherein the substrate nucleic acid strand comprises a 3′-end and a 5′-end and comprises a cytosine 2 to 6 nucleotides from the 3′-end of the substrate nucleic acid strand,wherein a nucleotide 2 to 6 nucleotides from the 5′-end of the substrate nucleic acid strand is replaced by an abasic site, andwherein the substrate nucleic acid strand hybridizes with the enzyme nucleic acid strand, resulting the substrate nucleic acid strand forming a molecular beacon comprising a stem region, wherein the stem region comprises the abasic site, wherein the abasic site is opposite to the cytosine 2 to 6 nucleotides from the 3′-end of the substrate nucleic acid strand. 19. The sensor of claim 18, wherein the stem region is 5 to 9 base pairs in length. 20. The sensor of claim 18, wherein the catalytic nucleic acid comprises a DNAzyme or an aptazyme. 21. The sensor of claim 1, wherein the sensor is attached to a solid support. 22. A kit comprising: the sensor of claim 1; andone or more of a buffer, a chart for correlating detected fluorescence and amount of target agent present, or a test agent. 23. A method for detecting a target agent, comprising contacting the sensor of claim 1 with a sample under conditions sufficient to allow the target agent in the sample to bind to the sensor resulting in cleavage of the sensor or a conformational change of the sensor; anddetecting fluorescence, wherein detection of fluorescence indicates the presence of the target agent in the sample, and an absence of detected fluorescence indicates the absence of the target agent in the sample. 24. The method of claim 23, further comprising quantifying the target agent, wherein a level of fluorescence detected indicates an amount of target agent present.
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