Aptamers against
Compositions comprising optimized aptamers capable of specifically binding to a surface protein of Clostridium difficile spore are provided. A method for detecting, enriching, separating, and/or isolating Clostridium difficile spores is provided.
1. A method of visualizing Clostridium difficile spores on a surface, comprising:
contacting a surface with a liquid composition comprising (a) at least one aptamer conjugated to a fluorophore, wherein the at least one aptamer has a stem-loop structure having a specific binding affinity for a surface protein of Clostridium difficile spore, wherein the at least one aptamer comprises a nucleic acid sequence having at least 90% identity with any one of the nucleic acid sequences as set forth in any of SEQ ID NOs: 43-55 wherein the surface protein is a spore coat surface protein or an exosporium layer protein; and (b) graphene oxide, wherein fluorophore is quenched by the association with the graphene oxide; and
visualizing the presence or absence of Clostridium difficile spores on the surface, wherein fluorescence is visible when the at least one aptamer is bound to the surface protein of Clostridium difficile spores.
2. The method of claim 1 , wherein when the at least one aptamer is not bound to the surface protein, the fluorophore is quenched and wherein when the aptamer binds to the surface protein, the fluorophore is not quenched.
3. The method of claim 1 , wherein the surface protein is CdeM, or CotE Chitinase.
4. The method of claim 1 , wherein the at least one aptamer comprises a nucleic acid sequence that selectively binds to surface protein CdeM of the Clostridium difficile spores, the aptamer having a stem-loop structure comprising Loop(3)/Stem(4f)/Loop(5)/Stem(4r)/Loop(5)/Stem(2f)/Loop(1)/Stem(2f)/Loop(1)/Stem(5 f)/Loop(5)/Stem(5r)/Loop(1)/Stem(2r)/Loop(1)/Stem(2r)/Loop(3).
5. The method of claim 1 , wherein the composition comprises two or more aptamers having a binding affinity to two or more epitopes of a surface protein of the Clostridium difficile spores or to two or more surface proteins of the Clostridium difficile spores.
6. The method of claim 1 , wherein the graphene oxide is in the form of nanoparticles.
7. The method of claim 1 , wherein the fluorophore emits at a wavelength of between about 510 nm and about 520 nm.
8. The method of claim 1 , further comprising illuminating the surface with a light source.
9. The method of claim 8 , wherein light from the light source has a predetermined wavelength, and the predetermined wavelength is different than a wavelength of light emitted by the fluorophore of the aptamer conjugate.
10. The method of claim 8 , wherein the light source is configured to produce light at a wavelength of between about 485 nm and about 515 nm.
11. The method of claim 8 , further comprising filtering the light produced by the light source such that light at a wavelength emitted by the fluorophore is visually detected.
12. The method of claim 8 , comprising passing the light produced from the light source through a bandpass filter such that light at a wavelength emitted by the fluorophore is visually detected.
13. The method of claim 8 , comprising passing the light produced from the light source through a circular polarizing filter such that light at a wavelength emitted by the fluorophore is visually detected.
14. The method of claim 1 , wherein the contacting comprises spraying.
15. A method of visualizing Clostridium difficile spores on a surface, comprising:
contacting a surface with a liquid composition comprising (a) at least one aptamer conjugated to a fluorophore, wherein the at least one aptamer has a stem-loop structure having a specific binding affinity for a surface protein of Clostridium difficile spore, wherein the at least one aptamer comprises a nucleic acid sequence as set forth in SEQ ID NO: 55, wherein the surface protein is a spore coat surface protein or an exosporium layer protein; and (b) graphene oxide, wherein fluorophore is quenched by the association with the graphene oxide; and
visualizing the presence or absence of Clostridium difficile spores on the surface, wherein fluorescence is visible when the at least one aptamer is bound to the surface protein of Clostridium difficile spores.
16. The method of claim 15 , wherein when the at least one aptamer is not bound to the surface protein, the fluorophore is quenched and wherein when the aptamer binds to the surface protein, the fluorophore is not quenched.
17. The method of claim 15 , wherein the at least one aptamer comprises a nucleic acid sequence that selectively binds to surface protein CdeM of the Clostridium difficile spores, the aptamer having a stem-loop structure comprising Loop(3)/Stem(4f)/Loop(5)/Stem(4r)/Loop(5)/Stem(2f)/Loop(1)/Stem(2f)/Loop(1)/Stem(5 f)/Loop(5)/Stem(5r)/Loop(1)/Stem(2r)/Loop(1)/Stem(2r)/Loop(3).
18. The method of claim 15 , wherein the composition comprises two or more aptamers having a binding affinity to two or more epitopes of a surface protein of the Clostridium difficile spores or to two or more surface proteins of the Clostridium difficile spores.
19. The method of claim 15 , wherein the graphene oxide is in the form of nanoparticles.
20. The method of claim 15 , wherein the fluorophore emits at a wavelength of between about 510 nm and about 520 nm.
21. The method of claim 15 , further comprising illuminating the surface with a light source.
22. The method of claim 21 , wherein light from the light source has a predetermined wavelength, and the predetermined wavelength is different than a wavelength of light emitted by the fluorophore of the aptamer conjugate.
23. The method of claim 21 , wherein the light source is configured to produce light at a wavelength of between about 485 nm and about 515 nm.
24. The method of claim 21 , further comprising filtering the light produced by the light source such that light at a wavelength emitted by the fluorophore is visually detected.
25. The method of claim 21 , comprising passing the light produced from the light source through a bandpass filter such that light at a wavelength emitted by the fluorophore is visually detected.
26. The method of claim 21 , comprising passing the light produced from the light source through a circular polarizing filter such that light at a wavelength emitted by the fluorophore is visually detected.