Method for isolating cross-reactive aptamer and use thereof
The subject invention provides a SELEX strategy for isolating cross-reactive aptamers that recognize a core structure of a small-molecule family and bind to each several structurally-similar molecules in the family. The subject invention also provides methods, assays, and products for detecting small-molecule targets of the family in a sample in both clinical and field settings. Such method is based on an aptamer sensor that reports the presence of small-molecule targets via a sensitive colorimetric signal for naked-eye detection. The subject invention further provides exonuclease-based methods for generating structure-switching aptamers from fully folded aptamers and developing electrochemical aptamer-based (E-AB) sensors for rapid and sensitive detection of synthetic cathinones.
1. An aptamer-based sensor comprising a cross-reactive aptamer for synthetic cathinones, the cross-reactive aptamer comprising SEQ ID NO: 17 or a sequence sharing at least 95% identity with SEQ ID No: 17.
2. The aptamer-based sensor according to claim 1 , the cross-reactive aptamer comprising SEQ ID No: 6, 18, or 19.
3. The aptamer-based sensor according to claim 1 , the cross-reactive aptamer being SEQ ID No: 6, 18, or 19.
4. The aptamer-based sensor according to claim 1 , the synthetic cathinone having a core structure of
wherein R 1 is selected from the group consisting of hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, alkenyl, alkynyl, alkoxy, haloalkyl, acyl, halogen, and hydroxylalkyl, or R 1 , taken together with the carbon atom to which it is attached and an adjacent carbon atom, form a substituted or unsubstituted 5- or 6-membered homocyclic or heterocyclic ring;
R 2 is hydrogen or alkyl; and
R 3 and R 4 are each independently selected from the group consisting of hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, alkenyl, alkynyl, haloalkyl, acyl, halogen, and hydroxylalkyl, or R 3 and R 4 , taken together with the nitrogen atom to which they are attached, form a substituted or unsubstituted 5- or 6-membered heterocyclic ring.
5. The aptamer-based sensor according to claim 4 , the synthetic cathinone being selected from 3,4-methylenedioxypyrovalerone (MDPV); α-PVP; pyrovalerone; methylone; pentylone; 3,4-methylenedioxy-α-pyrrolidinobutiophenone (MDPBP); mephedrone; 4-methyl-α-pyrrolidinobutiophenone (MPBP); 4′-methyl-α-pyrrolidinohexanophenone (MPHP); naphyrone; methedrone; ethylone; butylone; 4-methylmethcathinone (4-MMC); 4-fluoromethcathinone (4-FMC); 3-FMC; methcathinone; and 4-methyl-a-pyrrolidinobutiophenone (MEPBP).
6. The aptamer-based sensor according to claim 1 , the cross-reactive aptamer being modified with a reporter label selected from a fluorescent dye, a fluorescent or luminescent nanoparticle, and an affinity tag.
7. A method for detecting a synthetic cathinone in a sample, the method comprising contacting the sample with the aptamer-based sensor of claim 1 , the aptamer-based sensor further comprising a dye, and detecting the presence of the synthetic cathinone in the sample by a signal generated upon binding of the synthetic cathinone to the aptamer.
8. The method according to claim 7 , the dye being Cy7.
9. The method according to claim 7 , the sample being a biological sample or an environmental sample.
10. The method according to claim 9 , the biological sample being selected from blood, plasma, urine, tears, sweat, and saliva.
11. The method according to claim 7 , the cross-reactive aptamer comprising SEQ ID NO: 6, 18 or 19.
12. The method according to claim 7 , wherein the signal generated upon binding of the synthetic cathinone to the aptamer can be observed by the naked-eye.
13. The method according to claim 7 , the synthetic cathinone having a core structure of
wherein R 1 is selected from the group consisting of hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, alkenyl, alkynyl, alkoxy, haloalkyl, acyl, halogen, and hydroxylalkyl, or R 1 , taken together with the carbon atom to which it is attached and an adjacent carbon atom, form a substituted or unsubstituted 5- or 6-membered homocyclic or heterocyclic ring;
R 2 is hydrogen or alkyl; and
R 3 and R 4 are each independently selected from the group consisting of hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, alkenyl, alkynyl, haloalkyl, acyl, halogen, and hydroxylalkyl, or R 3 and R 4 , taken together with the nitrogen atom to which they are attached, form a substituted or unsubstituted 5- or 6-membered heterocyclic ring.
14. The method according to claim 13 , the synthetic cathinones being selected from MDPV; α-PVP; pyrovalerone; methylone; pentylone; MDPBP; mephedrone; MPBP; MPHP; naphyrone; methedrone; ethylone; butylone; 4-MMC; 4-FMC; 3-FMC; methcathinone; and MEPBP.