IPC분류정보
국가/구분 |
United States(US) Patent
공개
|
국제특허분류(IPC7판) |
|
출원번호 |
US-0961407
(2013-08-07)
|
공개번호 |
US-0051090
(2014-02-20)
|
우선권정보 |
EP-12180409.0 (2012-08-14) |
발명자
/ 주소 |
- Hulko, Michael
- Krasteva, Nadejda
- Hospach, Ingeborg
- Nelles, Gabriele
|
출원인 / 주소 |
|
인용정보 |
피인용 횟수 :
0 인용 특허 :
0 |
초록
▼
The present invention relates to aptazyme sensor devices comprising, in addition to the aptamer, ribozyme and communication components, a competitive inhibitory component with a metal nanoparticle or a competitive inhibitory and signalling component with a metal nanoparticle and a label, such that t
The present invention relates to aptazyme sensor devices comprising, in addition to the aptamer, ribozyme and communication components, a competitive inhibitory component with a metal nanoparticle or a competitive inhibitory and signalling component with a metal nanoparticle and a label, such that the enzymatic activity of the ribozyme is inhibited as long as the inhibitory or the inhibitory and signalling component is bound to the substrate binding site of the ribozyme. The aptazyme sensor devices of the invention have increased shelf-life and are suitable for the parallel detection of different ligands by using an array of aptazyme sensor devices utilizing inhibitory and inhibitory and signalling components with different metal nanoparticles. The present invention furthermore relates to the post-synthetic chemical modification of the aptamer component for avoiding unspecific binding.
대표청구항
▼
1. An aptazyme sensor device for detecting a ligand, said device comprising the following three components (a) an aptamer component, which comprises a binding site for a ligand,(b) a ribozyme component having a substrate binding site, wherein said ribozyme component has an enzymatic activity towards
1. An aptazyme sensor device for detecting a ligand, said device comprising the following three components (a) an aptamer component, which comprises a binding site for a ligand,(b) a ribozyme component having a substrate binding site, wherein said ribozyme component has an enzymatic activity towards a substrate, which enzymatic activity can be switched on and off, and which, if switched on, results in generation of a detectable signal upon exposure of the ribozyme component to the substrate and upon binding of the substrate to the substrate binding site,(c) a communication component, which is a single or double stranded nucleic acid and links (a) to (b) and translates changes in the binding state of component (a) to component (b),wherein (a), (b) and (c) are covalently bound, preferably in the order of (a), (c), (b) and/or in the order of (b), (c), (a), or combinations thereof, such as (b), (c), (a), (c), (b), or (a), (c), (b), (c), (a), furthermore comprising (d) an inhibitory component, which comprises a single nucleotide strand covalently bound to a metal nanoparticle, wherein the single nucleotide strand specifically and selectively binds to the substrate binding site of component (b) such that the enzymatic activity in (b) is inhibited,wherein binding of the single nucleotide strand to the substrate binding site of component (b) is selectively interrupted after exciting plasmon resonance of the metal nanoparticle by irradiation of said metal nanoparticle with light, wherein the enzymatic activity of component (b) is switched on when (i) a ligand is bound to component (a) and(ii) the inhibitory component (d) is not bound to component (b). 2. The aptazyme sensor device of claim 1, wherein component (a) comprises RNA, DNA, or combinations thereof. 3. The aptamer sensor device of claim 1 wherein component (a) is incorporated in component (b) and is flanked at either end by parts of components (c) and (b). 4. The aptazyme sensor device according to claim 1, furthermore comprising a substrate, which preferably is a substrate with a label, more preferably a molecular beacon or a scorpion primer, including a probe, or an aptazyme or riboswitch. 5. The aptazyme sensor device of claim 4, wherein the signal caused by said ribozyme component (b) results from the enzymatic activity of component (b) causing a change, preferably a cleavage of the substrate, preferably of the substrate with a label, causing the occurrence of said signal or of an increase in said signal. 6. The aptazyme sensor device of claim 1, wherein binding of the single nucleotide strand of inhibitory component (d) to the substrate binding site (of component (b)) is competitive. 7. The aptazyme sensor device of claim 1, wherein the single nucleotide strand of inhibitory component (d) comprises RNA, DNA, linker molecules, such as PEG, silanes, or combinations thereof. 8. The aptazyme sensor device of claim 1, wherein the metal nanoparticle of inhibitory component (d) is selected from gold, silver, palladium, copper, iron, nickel, titanium, bimetallic core-shell or heterodyne nanoparticles. 9. The aptazyme sensor device of claim 1, wherein component (c) comprises RNA, DNA, or combinations thereof, and wherein component (c) may occur several times within said aptazyme sensor device, e.g. if (a) is incorporated into (b) and is flanked by parts of (b) on either side. 10. The aptazyme sensor device of claim 1, wherein component (a) comprises chemical functionalities, e.g. oligoethylene side chains, carbohydrate chains, zwitterionic molecules, carboxy, amino, sulfide groups. 11. The aptazyme sensor device of claim 9, wherein component (a) comprises modified bases/nucleotides which comprise additional amino and/or carboxy and/or sulfide groups allowing reacting with carboxy groups and/or amine groups and/or maleinimide groups. 12. An array of aptazyme sensor devices for detecting different ligands comprising in one reaction vessel (i) a substrate with a label as defined in claim 4,(ii) at least two aptazyme sensor devices,wherein each aptazyme sensor device comprisesa different aptamer component (a) comprising a binding site for a different ligand, and an inhibitory component (d) comprising a different metal nanoparticle, such that, for each aptazyme sensor device and depending on the metal nanoparticle comprised, the binding of the inhibitory component (d) (to component (b)) is selectively interrupted by irradiation with light of a specific different wavelength. 13. The array of claim 12, wherein the reaction vessel is selected from a tube, a plate, a membrane, vessel, a slide, e.g. a microscopy slide, a porous material with an interstitial space for accommodating said array of aptazyme sensor devices, such as gels or polymers. 14. The array of claim 12, wherein the metal nanoparticles are immobilized onto a solid supportor are free nanoparticles, e.g. nanoparticles in solution. 15. An aptazyme sensor device comprising the following three components (a) an aptamer component, which comprises a binding site for a ligand,(b) a ribozyme component having a substrate binding site, wherein said ribozyme component has an enzymatic activity towards a substrate which enzymatic activity can be switched on and off,(c) a communication component, which is a single or double stranded nucleic acid and translates changes in the binding state of component (a) to component (b),wherein (a), (b) and (c) are covalently bound, preferably in the order of (a), (c), (b) and/or in the order of (b), (c), (a), or combinations thereof, such as (b), (c), (a), (c), (b), or (a), (c), (b), (c), (a), furthermore comprising (e) an inhibitory and signalling component, which comprises a single nucleotide strand having a ribozyme cleavage site and which has covalently bound a metal nanoparticle near or at the 5′ end and a label near or at the 3′ end yes or vice versa, wherein the single nucleotide strand binds to the substrate binding site of component (b) such that the enzymatic activity is inhibited,wherein after binding of the ligand to component (a) the enzymatic activity of component (b) is switched on and results in cleavage of the single nucleotide strand of component (e) at the cleavage site, but wherein no detectable signal is generated from the label,and wherein, after exciting plasmon resonance of the metal nanoparticle by irradiation with light, binding of the cleaved component (e) to component (b) is selectively interrupted such that the detectable signal is generated from the label. 16. The aptazyme sensor device of claim 15, wherein component (a) comprises RNA, DNA, or combinations thereof. 17. The aptazyme sensor device of claim 15, wherein the label is selected from fluorescent labels, luminescent labels, UV_Vis emitting or absorbing dyes, paramagnetic particles, paramagnetic particles having a fluorescent label attached, electrochemical labels, preferably fluorescent labels. 18-23. (canceled) 24. A method of detecting a ligand in a sample comprising (i) providing a sample,(ii) providing an aptazyme sensor device and a substrate with a label as defined in claim 4,(iii) contacting said sample with said aptazyme sensor device,(iv) irradiating the sample with said aptazyme sensor device with light of the wavelength which excites plasmon resonance of the metal nanoparticle of component (d) or (e) of the aptazyme sensor device,(v) detecting a signal. 25. A method of detecting different ligands in a sample comprising (i) providing a sample,(ii) providing an array of aptazyme sensor devices according to claim 12,(iii) contacting said sample with said array of aptazyme sensor devices,(iv) irradiating the sample with said array of aptazyme sensor devices with light of the wavelength which excites plasmon resonance of one type of metal nanoparticle of component (d) or (e) of the aptazyme sensor devices of said array,(v) detecting a signal,(vi) repeating steps (iv) and (v) depending on the number of different metal nanoparticles of said array of aptazyme sensor devices. 26. (canceled)
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