Compositions and methods for detecting pyrophosphate products of enzyme reactions using pyridylazoaniline dyes

Patent metadata

Field	Value
Publication number	US-11512342-B1
Application number	US-202217661954-A
Country	US
Kind code	B1
Filing date	May 4, 2022
Priority date	Nov 1, 2021
Publication date	Nov 29, 2022
Grant date	Nov 29, 2022

Abstract

Provided herein is a composition comprising an enzyme that releases pyrophosphate from a substrate and a dye of Formula 1. A method for detecting pyrophosphate is also provided. A kit comprising a polymerase that releases pyrophosphate by hydrolysis of nucleoside triphosphates during nucleic acid replication, a divalent manganese salt, and the dye are also provided. The present composition, method and kits provide a way to detect and/or quantify substrates or products of enzyme reacted substrates associated with the release pyrophosphate (e.g., nucleic acid amplification reactions and other reactions that hydrolyze ATP) via a distinct color change without substantially affecting the sensitivity and/or specificity of the reaction.

First claim

What is claimed is:

1. A composition, comprising; an enzyme that releases pyrophosphate from a substrate, a manganese ion (Mn 2+ ) and a dye of Formula 1:

wherein:
R1 and R5 are each independently selected from the group consisting of H, halogen, nitro, cyano, sulfonic acid, carboxy, trifluoromethyl, trichloromethyl and tribromomethyl;
R2 and R3 are each independently a lower alkyl optionally comprising a terminal sulfonate group; and
R4 is H, OH or COOH.

2. The composition of claim 1 , wherein:
at least one of R1 and R5 is F, Cl, Br or I;
R2 and R3 are each independently n-butyl, sec-butyl, isobutyl, tert-butyl, propyl, n-propyl, isopropyl, ethyl, or methyl and at least one of R2 and R3 comprises the terminal sulfonate group.

3. The composition according to claim 1 , wherein the composition comprises a manganese salt, wherein the molar ratio of the manganese salt to dye is greater than 1.

4. The composition according to claim 1 , wherein the composition further comprises one or more nucleoside triphosphates (NTPs) selected from rNTPs and dNTPs.

5. The composition according to claim 4 , wherein the molar ratio of NTPs to manganese salt is greater than 1.

6. The composition according to claim 1 , wherein the dye is 2-(5-Bromo-2-pyridylazo)-5-(N-propyl-N-sulfopropylamino)phenol (5-Br-PAPS) or 2-(5-Nitro-2-pyridylazo)-5-(N-propyl-N-sulfopropylamino)phenol (5-Nitro-PAPS).

7. The composition according to claim 1 , wherein the enzyme is selected from the group consisting of a DNA polymerase, RNA polymerase, reverse transcriptase, primase, ligase, helicase, nucleotide pyrophosphatase (Ppase)/phosphodiesterase, RNA decapping enzyme (RNA 5′ pyrophosphohydrolase) and geranyl pyrophosphate synthase.

8. The composition according to claim 1 , wherein the enzyme is a polymerase selected from the group consisting of a thermostable DNA polymerase, a strand displacing DNA polymerase, an RNA polymerase and a reverse transcriptase.

9. The composition according to claim 7 , wherein the enzyme is a DNA polymerase selected from the group consisting of Bst polymerase, Bsu polymerase, an archaeal DNA polymerase, Taq polymerase, or a variant thereof.

10. The composition according to claim 1 , wherein the composition is lyophilized or in an aqueous solution.

11. The composition according to claim 1 , further comprising a substrate for the enzyme, wherein cleavage of the substrate by the enzyme results in production of pyrophosphate.

12. The composition according to claim 1 , further comprising a nucleic acid sample that contains a target nucleic acid and, optionally, primers.

13. The composition according to claim 12 , wherein the sample is a sample from an animal subject.

14. The composition according to claim 12 , wherein the sample is a clinical sample selected from the group consisting of: saliva, nasal mucosa, urine, tissue biopsy, and a cell scrape or an environmental sample selected from the group consisting of water, sewerage, soil and plants or animal material for use as food.

15. A method for detecting pyrophosphate, comprising:
(a) combining a manganese salt, an enzyme, a substrate for the enzyme that releases pyrophosphate when it is cleaved by the enzyme, and a dye of Formula 1 produce a reaction mix;

wherein:
R1 and R5 are each independently selected from the group consisting of H, halogen, nitro, cyano, sulfonic acid, carboxy, trifluoromethyl, trichloromethyl and tribromomethyl;
R2 and R3 are each independently a lower alkyl optionally comprising a terminal sulfonate group; and
R4 is H, OH or COOH;
(b) incubating the reaction mix under conditions by which the enzyme cleaves the substrate to produce pyrophosphate; and
(c) observing a change in color of the reaction mix, wherein the change in color indicates production of pyrophosphate.

16. The method of claim 15 , wherein the enzyme is selected from the group consisting of a DNA polymerase, an RNA polymerase, a reverse transcriptase, a primase, a ligase, a helicase, a nucleotide pyrophosphatase (Ppase)/phosphodiesterase, an RNA decapping enzyme (RNA 5′ pyrophosphohydrolase) and a geranyl pyrophosphate synthase.

17. The method of claim 15 , wherein the substrate is an NTP.

18. The method of claim 15 , wherein:
the reaction mix comprises a polymerase, rNTPs or dNTPs, the manganese salt, a template nucleic acid and the dye;
the reaction mix is incubated under conditions suitable for amplification of the nucleic acid; and
the change in color indicates that a product has been amplified.

19. The method of claim 18 , wherein the reaction mix comprises an amplification mix for loop-mediated isothermal amplification (LAMP), polymerase chain reaction (PCR), strand displacement amplification (SDA), helicase dependent amplification (HDA), multiple displacement amplification (MDA), rolling circle amplification, ligation mediated amplification, whole genome amplification (WGA), nucleic acid sequence-based amplification (NASBA) or recombinase polymerase amplification (RPA) mix.

20. The method according to claim 18 , wherein the reaction mix is a PCR reaction mix and the method comprises thermocycling the reaction mix.

21. The method according to claim 18 , wherein reaction mix is a LAMP reaction mix and the method comprises incubating the reaction mix under isothermal conditions.

22. The method according to claim 15 , wherein the color change in (c) is red to yellow wherein yellow indicates production of pyrophosphate.

23. The method according to claim 15 , wherein (c) further comprises detecting the color change by spectrophotometry, image analysis or by eye.

24. The method according to claim 15 , wherein (c) further comprises detecting the color change by a change in hue or a change in peak wavelength.

25. The method according to claim 15 , wherein the change in color is detected by observing a change in a ratio of wavelength peak heights, before and after step (b).

26. The method according to claim 25 , wherein the wavelength peak heights are in the range of 350 nm-620 nm, 430 nm-480 nm and/or 520 nm-620 nm.

27. The method according to claim 18 , further comprising, determining the amount of nucleic acid substrate or the amount of amplicon by determining the number of thermocycles in a PCR amplification reaction for producing a predetermined ratio of wavelength peak height.

28. The method according to claim 15 , wherein the dye is 2-(5-Bromo-2-pyridylazo)-5-(N-propyl-N-sulfopropylamino)phenol (5-Br-PAPS) or 2-(5-Nitro-2-pyridylazo)-5-(N-propyl-N-sulfopropylamino)phenol (5-Nitro-PAPS).

29. A kit comprising a polymerase, manganese salt, and a dye of formula 1

wherein:
R1 and R5 are each independently selected from the group consisting of H, halogen, nitro, cyano, sulfonic acid, carboxy, trifluoromethyl, trichloromethyl and tribromomethyl;
R2 and R3 are each independently a lower alkyl optionally comprising a terminal sulfonate group; and
R4 is H, OH or COOH.

Assignees

• New England Biolabs Inc

Inventors

• Tanner Nathan
• Correa Jr Ivan R
• Zhang Yinhua
• Alpaslan Ece

Classifications

• C12Q1/6816 Primary

  characterised by the detection means (C12Q1/6804 takes precedence) · CPC title

• C12Q1/6876

  Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes · CPC title

• C12Q1/6846

  Common amplification features · CPC title

• G01N33/84

  involving inorganic compounds or pH · CPC title

• C12Q1/48 Primary

  involving transferase · CPC title