Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Visual and modular detection of nucleic acids with enzyme-assisted nanotechnology

A nucleic acid and target nucleic acid technology, applied in the field of nucleic acid detection, can solve problems such as high cost, expensive usage of probes, and complexity

Pending Publication Date: 2021-06-25
NAT UNIV OF SINGAPORE +1
View PDF2 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Alternatively, sequence-specific signaling probes (such as fluorescent Taqman reporters) can be used to improve detection accuracy; however, these probes are expensive and complex to use [Gardner, S.N. et al., J Clin Microbiol 41:2417-2427 (2003)]
As dedicated sequence-specific probes are required for each stretch of DNA target for coupled signaling during target amplification, the methods become increasingly costly and difficult to multiplex or perform complex calculations [Juskowiak, B.Anal Bioanal Chem 399:3157-3176 (2011)]

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Visual and modular detection of nucleic acids with enzyme-assisted nanotechnology
  • Visual and modular detection of nucleic acids with enzyme-assisted nanotechnology
  • Visual and modular detection of nucleic acids with enzyme-assisted nanotechnology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0123] method

[0124] Identifying Nanostructure Characterization

[0125] All sequences can be found in Tables 1-7 and were purchased from Integrated DNA Technologies (IDT). To prepare recognition nanostructures, in 50mM NaCl, 1.5mM MgCl 2 and 50mM Tris-HCl buffer (pH 8.5) were mixed with DNA aptamer and reverse body oligonucleotides in equimolar ratio. The mixture was incubated at 95°C for 5 minutes and cooled slowly at 0.1°C / s until the reaction reached 25°C, after which Taq DNA polymerase ( Promega) to form a hybrid complex. To characterize the assembly and activity of the recognition nanostructures in the presence of the DNA target, different concentrations of the target oligonucleotides were added to this mixture. The real-time association and dissociation kinetics of the complexes were measured by biolayer interferometry (Pall Fortebio). Briefly, preassembled biotin-aptamers were immobilized on streptavidin-functionalized interferometric sensors. After a brief wa...

Embodiment 2

[0168] enVision platform

[0169] The enVision platform consists of a series of enzyme-assisted DNA nanostructures to enable three functional steps: DNA target recognition, target-independent signal enhancement, and visual detection ( figure 1 a). Target recognition is decoupled from signal enhancement through orthogonal sequence design of nanostructures. In the recognition step, the recognition element is a unique hybrid nanostructure. It consists of a modified aptamer bound to a TaqDNA polymer [Dang, C. & Jayasena, S.D.J Mol Biol 264:268-278 (1996); Park, K.S. et al. Sci Adv 2:e1600300 (2016)]. In the absence of target DNA, the aptamer binds firmly to the polymerase to inhibit the polymerase activity. Following target hybridization in the presence of complementary target DNA, the hybrid dissociates to activate polymerase activity. During the signaling step, the active polymerase elongates the universal self-initiating nanostructure in a target-independent manner. By inc...

Embodiment 3

[0172] Optimized Assay for Visual Quantification of Nucleic Acids

[0173] The performance of DNA nanostructures as functional recognition and signaling elements were evaluated, respectively. For recognition of nanostructures, polymerases associate with modified aptamers to form hybrid complexes ( figure 2 a, above). This optimized complex exhibited negligible polymerase activity ( Figure 8 a). In the presence of complementary target DNA, the hybrid structure dissociates and polymerase activity is fully restored ( figure 2 a, the figure below). This recognition and activation was found to exhibit high sequence specificity, as only complementary targets resulted in strong polymerase activity, whereas scrambled oligonucleotide sequences produced negligible activity ( Figure 8 b).

[0174] The universal recognition element is further designed as a self-initiated nanostructure. The structure exhibits enhanced polymerase occupancy and activity compared to its similarly s...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The present invention provides methods and devices for specific detection of nucleic acids using an integrated circuit of two independent enzyme-DNA nanostructures - an easily adjustable recognition element and a sensitive universal signaling element - to decouple target recognition and visual signal amplification. The recognition element comprises a DNA polymerase enzyme, a DNA polymerase enzyme-specific DNA aptamer and an inverter oligonucleotide. In the presence of a target nucleic acid, the inverter oligonucleotide binds to the target nucleic acid and releases the DNA polymerase enzyme from inhibition by the DNA aptamer. The activated DNA polymerase enzyme is then contacted with a signaling nanostructure comprising a self-priming portion responsive to the DNA polymerase enzyme, in the presence of labelled dNTPs and signal development reagents, wherein the activated DNA polymerase enzyme would add the labelled dNTPs to the self-priming portion, followed by the binding of the signal development reagents to the labelled dNTPs.

Description

technical field [0001] The present invention relates to nucleic acid detection using enzyme-assisted nanotechnology. More specifically, the present invention provides methods and devices for the specific detection of nucleic acids using an integrated circuit of two independent enzyme-DNA nanostructures (i.e., an easily tunable recognition element and a sensitive general-purpose signaling element) , to decouple target recognition from visual signal amplification. Background technique [0002] Detection of pathogenic nucleic acids has broad applications in infection diagnosis and management. As an alternative to routine pathogen culture that requires long processing times (i.e., days) and requires species-specific protocols (e.g., bacteria vs. and beyond) unprecedented molecular information on infection [Niemz, A., Ferguson, T.M. and Boyle, D.S. Trends Biotechnol 29:240-250 (2011); Nong, R.Y. et al., Expert Rev Proteomics 9:21-32 (2012) ; Zumla, A. et al. Lancet Infect Dis ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/682
CPCC12Q1/6844C12Q1/708B01L2400/049B01L2200/04B01L2300/0883B01L2300/0825B01L2200/16C12Q2521/101C12Q2525/205C12Q2563/107C12Q2563/149C12Q2565/629C12Q1/701B01L2200/0663C12M23/16C12M23/42C12N15/115C12N2310/16C12Q1/682
Inventor 邵慧琳何瑞原
Owner NAT UNIV OF SINGAPORE
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com