Method for isolating cross-reactive aptamer and use thereof

Patent metadata

Field	Value
Publication number	US-10907162-B2
Application number	US-202016791101-A
Country	US
Kind code	B2
Filing date	Feb 14, 2020
Priority date	Oct 30, 2018
Publication date	Feb 2, 2021
Grant date	Feb 2, 2021

Abstract

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.

First claim

We claim:

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.

Assignees

• Yang Weijuan
• Yu Haixiang
• Liu Yingzhu
• Xiao Yi
• The Florida International Univ Board Of Trustees

Inventors

• Yang Weijuan
• Yu Haixiang
• Liu Yingzhu
• Xiao Yi

Classifications

• C12N2330/31

  Libraries, arrays · CPC title

• A61K31/137

  Arylalkylamines, e.g. amphetamine, epinephrine, salbutamol, ephedrine {or methadone} · CPC title

• C12N15/115 Primary

  Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith {; Nucleic acids binding to non-nucleic acids, e.g. aptamers} · CPC title

• C12N2310/16

  Aptamers · CPC title

• A61K31/167 Primary

  having the nitrogen of a carboxamide group directly attached to the aromatic ring, e.g. lidocaine, paracetamol · CPC title