Nucleic acid-induced aggregation of metal nanoparticles and uses thereof in methods for detecting nucleic acids

Patent metadata

Field	Value
Publication number	US-2017335376-A1
Application number	US-201515521165-A
Country	US
Kind code	A1
Filing date	Oct 23, 2015
Priority date	Oct 23, 2014
Publication date	Nov 23, 2017
Grant date	—

Abstract

The invention relates to an aggregate comprising metallic nanoparticles and nucleic acid molecules wherein each metallic nanoparticle is coated with a polycation. The invention also relates to a method for obtaining the aggregate of the invention and to the use of said aggregate in methods for detecting the presence of a nucleic acid in a sample, in methods for detecting the presence of a given nucleotide at a predetermined position in a target nucleic acid, in methods for detecting the presence of a modified nucleotide at a predetermined position in a target nucleic acid, methods for detecting the presence of a conjugate between a double stranded nucleic acid and a chemical in a sample comprising double stranded nucleic acid molecules, in methods for determining the content of modified nucleotides in a target nucleic acid and in a method for determining the content of modified nucleotides in a target nucleic acid.

First claim

1 .- 49 . (canceled)

50 . An aggregate comprising metallic nanoparticles and nucleic acid molecules wherein each metallic nanoparticle is coated with a polycation and wherein said aggregate is formed by electrostatic interactions between the negative charges in the nucleic acid molecules and the positive charges of the polycation in the coats of said metallic nanoparticles, wherein the polycation is selected from the group consisting of ethylene diamine, 1,3-diaminopropane, hexamethylenediamine, putrescine and cadaverine.

51 . The aggregate according to claim 50 wherein the metal is silver, gold or a combination thereof.

52 . The aggregate according to claim 50 , wherein the polycation is putrescine.

53 . The aggregate according to claim 50 , wherein the nucleic acid is selected from the group consisting of RNA, DNA, a double stranded nucleic acid, a single stranded nucleic acid, methylated DNA, a coordination complex of a nucleic acid and a metal, a coordination complex of a nucleic acid and a compound containing a metal and a complex of a nucleic acid and an intercalating organic dye.

54 . A method for obtaining an aggregate according to claim 50 comprising the steps of:
(i) obtaining a population of metallic nanoparticles by contacting a salt of a metal and a hydrochloride of a polycation in the presence of a reducing agent under conditions adequate for the formation of the metallic nanoparticles coated with said polycation; and
(ii) contacting the nanoparticles obtained in step (i) with a nucleic acid under conditions adequate for the formation of an aggregate formed by electrostatic interaction between a negatively charged nucleic acid and the positive charges of the polycation in the coat of said metallic nanoparticles.

55 . A method selected from the group consisting of:
(A) A method for detecting the presence of a nucleic acid in a sample, comprising the steps of: a. contacting said sample with a population of metallic nanoparticles, wherein said metallic nanoparticles are coated with a polycation thereby forming aggregates of said metallic nanoparticles stabilized by electrostatic interactions between the negative charges in the nucleic acid and the positive charges of the polycation; and b. obtaining a SERS spectrum of the sample wherein an increase in the SERS spectrum of a band characteristic of a purine or pyrimidine base in a nucleic acid forming part of the aggregate is indicative of the presence of a nucleic acid in the sample and wherein I. if the band is selected from the group consisting of a band at about 503 cm −1 , at about 621 cm −1 , at about 665/677 cm −1 , at about 730 cm −1 , at about 752 cm −1 , at about 787 cm −1 , at about 1019 cm −1 , at about 1324 cm −1 , at about 1653 cm −1 , at about 2806 cm −1 and at about 2967 cm −1 , then the nucleic acid is double stranded DNA, II. if the band is selected from the group consisting of a band at about 512 cm −1 , about 686 cm −1 , at about 734 cm −1 , at about 793 cm −1 , at about 1029 cm −1 , at about 1199 cm −1 , at about 1329 cm −1 , at about 1643 cm −1 and at about 2960 cm −1 , then the nucleic acid is single stranded DNA or III. if the band is selected from the group consisting of a band at about 599 cm −1 , at about 1090 cm −1 , at about 1178 cm −1 , at about 1246/1264 cm −1 , at about 1354 cm −1 , at about 1376 cm −1 , at about 1421 cm −1 , at about 1487 cm −1 , at about 1509 cm −1 , at about 1528 cm −1 , at about 1577 cm −1 and at about 1628 cm −1 , then the nucleic acid is single stranded RNA or double stranded RNA; (B) A method for detecting the presence of a given nucleotide at a predetermined position in a target nucleic acid comprising the steps of: (i) contacting a population of metallic nanoparticles coated with a polycation separately with the target nucleic acid and with a control nucleic acid having the same sequence as the target nucleic acid and having a known nucleotide at said predetermined position, thereby resulting in the formation of a first type of aggregates comprising the metallic nanoparticles and the target nucleic acid and a second type of aggregates comprising the metallic nanoparticles and the control nucleic acid, (ii) obtaining the SERS spectra of the first and second types of aggregates obtained in step (i); and wherein if the SERS spectrum of the first type of aggregates and the SERS spectrum of the second type of aggregates are substantially identical, then the nucleotide at said predetermined position in the target nucleic acid is the same as the known nucleotide or wherein if the SERS spectrum of the first type of aggregates and the SERS spectrum of the second type of aggregates are the SERS spectrum are different, then the nucleotide at said predetermined position is different from the known nucleotide; (C) A method for detecting the presence of a modified nucleotide at a predetermined position in a target nucleic acid comprising the steps of: (i) contacting a population of metallic nanoparticles coated with a polycation separately with the target nucleic acid and with a control nucleic acid having the same sequence as the target nucleic acid and wherein the predetermined position is not modified, thereby resulting in the formation of a first type of aggregates comprising the metallic nanoparticles and the target nucleic acid and a second type of aggregates comprising the metallic nanoparticles and the control nucleic acid, (ii) obtaining the SERS spectra of the first and second types of aggregates obtained in step (i) and wherein if the SERS spectrum of the first type of aggregates and the SERS spectrum of the second type of aggregates are substantially identical, then the nucleotide at said predetermined position is not modified or wherein if the SERS spectrum of the first type of aggregates and the SERS spectrum of the second type of aggregates are the SERS spectrum are different, then the nucleotide at said predetermined position is modified; and (D) A method for detecting the presence of a conjugate between a double stranded nucleic acid and a chemical in a sample comprising double stranded nucleic acid molecules comprising the steps of: (i) contacting said sample with a population of metallic nanoparticles coated with a polycation, thereby forming an aggregate comprising metallic nanoparticles coated with a polycation and double stranded nucleic acid molecules stabilized by electrostatic interactions between the negative charges in the nucleic acid molecules and the positive charges of the polycation; and (ii) obtaining the SERS spectrum of said sample, wherein the presence in the spectrum of a one or more bands characteristic of the interaction between the nucleic acid and the chemical or of the chemical is indicative of the presence of said conjugate in the sample.

56 . The method according to claim 55 (B) wherein
a. if the difference between the spectra of the first and second types of aggregates is an increase in the intensity of a band selected from the group consisting of a band at 730 cm −1 , at 734 cm −1 , at 1224 cm −1 , at 1329 cm −1 , at 1508 cm −1 and 1577 cm −1 , then it is indicative that the nucleotide at said predetermined position is adenine, b. if the difference between the spectra of the first and second types of aggregates is an increase in the intensity of a band at 1577 cm −1 , then it is indicative that the nucleotide at said predetermined position is adenine or guanine, c. if the difference between the spectra of the first and second types of aggregates is an increase in the intensity of a band selected from the group consisting of a band at 621 cm −1 , at 665/677 cm −1 , at 686 cm −1 , at 1354 cm −1 , at 1487 cm −1 , then it is indicative that the nucleotide at said predetermined position is gu

Assignees

• Univ Rovira I Virgili
• Institució Catalana De Recerca I Estudis Avançats
• Fundació Centre Tecnològic De La Química De Catalunya
• Univ Strathclyde
• Medcom Tech S A

Inventors

• Álvarez Puebla Ramón Ángel
• Guerrini Luca
• Sagalés Mañas Juan
• Graham Duncan

Classifications

• C12Q2565/632

  being a surface enhanced, e.g. resonance, Raman spectrometer · CPC title

• B82Y15/00

  Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors · CPC title

• B82Y5/00

  Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery · CPC title

• C12Q2563/155

  Particles of a defined size, e.g. nanoparticles · CPC title

• C12Q1/6816 Primary

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