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Sequence-selective recognition of nucleic acids using nanoparticle probes

A technology of nanoparticle and target nucleic acid, applied in nanotechnology for sensing, nanotechnology, nanotechnology, etc., which can solve the problems of difficult preparation and low solubility of PNA/nanoparticle conjugates

Active Publication Date: 2013-01-16
AGENCY FOR SCI TECH & RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the low solubility of PNA oligomers in aqueous solution makes the preparation of PNA / nanoparticle conjugates extremely difficult

Method used

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  • Sequence-selective recognition of nucleic acids using nanoparticle probes
  • Sequence-selective recognition of nucleic acids using nanoparticle probes
  • Sequence-selective recognition of nucleic acids using nanoparticle probes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0119] Example 1: Materials

[0120] Chemical reagent: tetrachloroauric(III) acid trihydrate (HAuCl 4 ·3H 2 O), trisodium citrate, TritonX-100, sodium dodecyl sulfate (SDS), ZonylFSN-100 (F(CF 2 CF 2 ) 3-8 CH 2 CH 2 O(CH 2 CH 2 O) 0-15 H), tris(2-carboxyethyl)phosphine (TCEP) and dithiothreitol (DTT) were purchased from Sigma-Aldrich (St Louis, MO). 40-nm silver nanoparticles were purchased from TedPella (Redding, CA). All other reagents certified as analytically pure were used as received. Morpholino oligomers were purchased from GeneTools LLC (Philomath, OR). oligonucleotides from 1 st Acquired from Base Pte Ltd (Singapore).

[0121] Instrument: with NanoDrop TM A 1000 spectrophotometer (Thermo Scientific) was used for quantification of morpholino oligonucleotides and ssDNA samples. Agilent G1103AUV-Vis spectrophotometer was used to collect the absorption spectrum of gold nanoparticle colloid. This spectrophotometer was also used for melting analysis. Absorb...

Embodiment 2

[0122] Example 2: Synthesis of gold nanoparticles

[0123] By reducing HAuCl with citrate as described by Grabar, K.C. et al., Anal.Chem., 1995, 67, 735-743 4 to prepare gold nanoparticles with average diameters of ~40 nm and ~13 nm, respectively. All glassware used to prepare gold nanoparticles was sequentially filled with freshly prepared aqua regia (HNO 3 :HCl=1:3), rinse thoroughly with ultrapure water, and then dry in an oven at 100°C for 2-3 hours. 60 mL of 0.01% (w / v) HAuCl 4 Boil in a round bottom flask with reflux condenser and stir vigorously. For gold nanoparticles with an average diameter of ~40 nm, add 0.6 mL of 1.0% (w / v) sodium citrate to HAuCl 4 solution. For gold nanoparticles with an average diameter of ~13 nm, add 4.5 mL of 1.0 wt% sodium citrate to HAuCl 4 in solution. The reaction mixture was maintained at boiling point and stirring was continued for about 15 minutes. The suspension was stored at 4°C until subsequent use. Assuming spherical partic...

Embodiment 3

[0124] Example 3: Preparation of metal nanoparticles modified with mercapto-morpholino oligonucleotides

[0125]By activating the sulfhydryl group, the 3'-disulfamide-modified MO was treated with 0.1 MDTT in 0.2M phosphate buffered saline (PBS, pH 8.0) for 1 hour; the thiolated MO was then purified using a NAP-5 column (GE Healthcare). The purified thiolated MO samples were stored at 4°C until subsequent use. To avoid interchain disulfide formation of the thiolated MO chains, the purified MO was dissolved in 5 mM tris(2-carboxyethyl)phosphine (TCEP) (pH 7.5) before mixing with gold or silver nanoparticles. Incubate for 10 minutes. Unless otherwise stated, the mixture solution containing ˜2 μM thiolated MO, ˜2 nM or 4 nM FSN-capped nanoparticles, ˜0.1 wt % SDS, and 10 mM phosphate buffer (pH 7.5) was incubated at room temperature for 1 h or 2 h. Hour. Then, excess ssDNA was removed by centrifugation at 7.0 Krpm for 10 minutes. Unreacted MO was removed by centrifugation at 1...

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Abstract

The present invention refers to a conjugate comprising a nanoparticle and at least one oligonucleotide analog, wherein the at least one oligonucleotide analog is a phosphorodiamidate morpholino oligo (PMO) or a derivative thereof that is covalently coupled to the nanoparticle, methods for detecting a target nucleic acid using the conjugate and a kit comprising the conjugate.

Description

[0001] Cross References to Related Applications [0002] This application claims priority from Singapore Patent Application No. 201000176-6 filed on 12 January 2010, the contents of which are hereby incorporated by reference in their entirety for all purposes. technical field [0003] The present invention relates to conjugates for nucleic acid detection, said conjugates comprising nanoparticles and at least one oligonucleotide analogue, methods of using the conjugates, uses of the conjugates and methods comprising the present invention Kit of at least one conjugate. Background technique [0004] In life science research, nucleic acid detection using hybridization-based techniques is widely used. Sequence-specific detection of DNA has always been one of the focuses of biological analysis. Identification of single nucleotide variants is critical in many research areas, such as genotyping of single nucleotide polymorphisms (SNPs) and detection of acquired point mutations. S...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C07D413/00G01N21/31B82Y15/00C07H21/00
CPCC07D413/04B82Y15/00C07D487/04C12Q1/6827C12Q2563/155C12Q2563/137C12Q2527/107
Inventor 祖延兵高志强
Owner AGENCY FOR SCI TECH & RES
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