Detecţia multiplă şi ultra-senzitivă a fragmentelor scurte de acizi nucleici, utilizând nanoparticule de aur şi nanopori proteici

Nanopore-based, ultra-sensitive and multivalent detection of short nucleic acid fragments, with functionalized gold nanoparticles

-NANOSENSEDNA –

Obiectivul general al proiectului constă în identificarea unui sistem extrem de selectiv de detectare a fragmentelor scurte de acizi nucleici monocatenari (ssDNA) în probe biologice cu concentrații relevante fiziologic, adecvat și pentru recunoașterea în timp real a mutațiilor unei singure nucleotide în secvența ssDNA.

The core purpose of this proposal is to discover knowledge and thus lay the foundation of a new paradigm toward ssDNA detection, with SNV capability, enabling accurate distinguishing between closely related sequences – different by as few as one base, working in buffer as well as in biological samples, showing sensitivity to biologically-relevant concentrations.

Abstract: Identificarea rapidă, ieftină și precisă a fragmentelor scurte de acizi nucleici monocatenari (ssDNA) endogene sau exogene, permite depistarea precoce a diferitelor boli şi identificarea prezenței agenților patogeni virali sau microbieni. În acest proiect propunem dezvoltarea unui sistem extrem de selectiv de detectare a ssDNA în probe biologice, adecvat și pentru recunoașterea în timp real a mutațiilor unei singure nucleotide în secvența ssDNA. Implementând metode din domeniul nanotehnologiei bazate pe nanopori proteici și nanoparticule de aur (AuNPs) funcționalizate cu acizi nucleici, urmărim: i) detecţia analiţilor cu ajutorul nanoporilor proteici şi ii) utilizarea acizilor nucleici peptidici (PNA) ca ținte extrem de specifice pentru captarea și recunoașterea ssDNA, prin procese de hibridizare specifică, datorită afinităţii și selectivităţii mai mari ai PNA pentru ssDNA. Astfel, utilizând sistemul nanopor-AuNP-PNA ca transductor molecular pentru detecţia individuală a ssDNA, vom obţine un grad ridicat de specificitate şi sensibilitate pentru identificarea în timp real a ssDNA, bazându-ne pe: i) utilizarea AuNPs funcţionalizate cu monocatene de PNA cu sau fără grupări terminale tiol, ca ţinte pt detecţia multivalentă a unor ssDNA particulare, contribuind astfel la o îmbunătăţire a limitei de detecţie şi ii) sustenabilitatea tehnicii prezentate pentru detecţia şi identificarea unimoleculară. Beneficiile sociale ale acestui proiect constau în dezvoltarea în viitor a medicinei personalizate, bazată pe profilul unic de boală extras din ADN și implementarea facilă a testelor în timp real de analiză a ssDNA din sistemul circulator şi a probelor lichide de biopsie. Integrarea nanotehnologiilor şi a rețelelor neuronale artificiale, pentru a extrage automat informații unimoleculare cu ajutorul sitemului nanopor-AuNP-PNA de detecţie a ssDNA, detaliat în această propunere, oferă o nouă metodă pentru detectarea multiplă a mai multor biomarkeri ssDNA, într-o singură execuție.

Abstract: Fast, cheap and accurate detection of endogenous or exogenous short DNA fragments, may enable early detection of various diseases or probe the presence of viral or microbial pathogens. In this proposal we aim at integrating two synergic nanotechnology tools, namely nucleic acids-functionalized gold nanoparticles (AuNPs) and nanopores, to provide a highly selective and sensitive ssDNA detection system in aqueous samples, also suitable for real-time recognition of single-nucleotide variations (SNV). To this end, the strategy involves: (i) protein nanopores-based analytes biosensing, and (ii) the use of peptide-nucleic acids (PNA) probes to serve at highly specific targets for ssDNA capture and recognition, via specific hybridization. The combined use of a PNA-nanopore system, as a single-molecule transducer of ssDNA detection, will provide exquisite specificity and sensitivity for real-time, label-free ssDNA detection. The specificity attribute arises as PNAs present greater binding affinity and selectivity versus conventional nucleic acid probes. The unsurpassed sensitivity of detection is imparted by: (i) the use of AuNPs functionalized with free- and thiol-terminated single stranded PNAs, targeted for multi-valent detection of specific ssDNAs, thus contributing to the lower ssDNA detection limit, and (ii) the capability of the presented platform for single particle detection and discrimination. The social benefits of this project lie, among others, on the future development of personalized care, provided based on the unique nucleic acid-entailed disease profile, and the facile implementation of real-time, cell-free circulating DNAs, liquid-biopsy-based assays. Integrating nanomanufacturing technologies combined with machine learning-based convolutional neural networks, to automatically extract single-molecule information from the ssDNA detection platform revealed herein, may provide a boosting to the multiplexed detection of multiple ssDNA biomarkers in a single run.

The specific objectives and activities to be implemented, are as follows:

Objective I.  Gold nanoparticles (AuNPs) as a suitable matrix for binding non-functionalized and peptide-functionalized PNAs.

A I.1. Fabrication of PNA (free- and thiol-based) – conjugated AuNP. Colorimetric and UV-VIS Spectroscopy Assessment of PNA-Induced AuNP Aggregation. AuNPs size-dependent PNA binding (months 1-3).

A I.2. Assessment of probe PNA (free- and thiol-based) affinity and immobilization efficiency onto the AuNPs, based on the nature of PNA, size, thiol molecule, linker size and charge. Stabilization effects of electrolyte (ionic strength, pH) on the probe PNA-AuNP interactions (months 2-6).

A I.3. Optimization of electrolyte buffer for nanopore analysis. Nanopore measurement, statistical assessment and quantification of AuNP aggregation dynamics and capture at the nanopore entrance, before and after PNA conjugation (months 5-9).

Objective II. Target detection of ssDNAs via hybridization with AuNP-PNA conjugate constructs.

A II.1. Colorimetric and calorimetric assessment of target ssDNAs adsorption-mediated interaction with non-conjugated AuNPs (months 9-12).

A II.2. Quantification of target ssDNA detection by free- and thiol-functionalized PNA-AuNP conjugates. Comparative thermodynamics analysis of DNA hybridization on free- and thiol-functionalized PNA-AuNP conjugates (months 10-13).

A II.3. Influence of ssDNA hybridization on PNA-AuNP conjugates aggregation stability, based on AuNP size, PNA type (free or thiol-functionalized), salt, pH (months 10-15).

A II.4. Nanoparticle aggregation/dispersion-based, selective detection of single-nucleotide variations (SNV) and multiple mismatches on similarly sized ssDNAs, using PNA-conjugated AuNPs (months 11-15).

Objective III. Non-amplified, quantitative assessment of ssDNA sequences detection with single-protein nanopores, using the hybridization with a PNA-AuNP probe set.

A III.1. Single, PNA-free and PNA-conjugated AuNP detection with nanopores. Quantitative estimators probing the stochastic fingerprint of AuNP-nanopore interactions (months 15-20).

A III.2. Nanopore-based detection of target ssDNA in electrolyte buffers, via hybridization with PNA-conjugated AuNP systems (months 18-24).

A III.3. Multiplexed, selective single-molecule sensing of target ssDNAs in complex mixtures and assessment of SNVs presence multiple mismatches, via hybridization with PNA-conjugated AuNPs. Nanopore-based kinetic analysis of ssDNA–(PNA-AuNP) hybridization interactions (months 18-24).

Echipa proiectului/The project team:

  • Loredana-Cristina MEREUTA (Director proiect/The Project leader)
  • Dr. Alina Asandei [Major area of expertise regards investigation of molecules interaction with protein pores and peptide conformational changes at the single-molecule level using a protein nanopore.]
  • Dr. Irina Schiopu [Research abilities revolve around stochastic sensing with nanopores and analytical techniques regarding membrane biology, UV-VIS and fluorescence spectroscopy techniques applied in the study of customize peptides, dendrimers and DNA/PNA molecules.]
  • Ph.D. Isabela Dragomir [is proficient with electrophysiology techniques, biochemistry techniques related to membrane reconstitution and she is trained in isothermal calorimetry techniques.]

The social benefits. SNV disease-specific discoveries are hoped to propel to the development of personalized medicine, wherein optimal care is provided to individuals depending on their unique disease profile. For example, such tools require no specialized infrastructure or skilled personnel to perform the analysis, rendering them easily translatable to sites with low-resource settings. Another appealing potential application for the discoveries envisioned herein, is represented by real-time, liquid-biopsy-based SNV assays, useful in the diagnosis and monitoring of cancer, through the analysis of cell-free circulating tumor DNA (ctDNA).Fast, accurate and economically viable analysis ofsuch blood (liquid biopsy)-based, tumor-derived genetic materials, presents a minimal risk to the patient and may permit early detection of pathologies long before solid tumor become detectable.

Dissemination of scientific results:

 1.Article: 

  • Sequence-specific detection of single-stranded DNA with a gold nanoparticle-protein nanopore approach, By: Mereuta, L (Mereuta, Loredana)1 ] Asandei, A (Asandei, Alina)2 ] Dragomir, IS (Dragomir, Isabela S.)2 ] Bucataru, IC (Bucataru, Ioana C.)1 ] Park, J (Park, Jonggwan)3 ] Seo, CH (Seo, Chang Ho)3 ] Park, Y (Park, Yoonkyung)4,5 ] Luchian, T (Luchian, Tudor)1 ]; Scientific Reports (http://www.nature.com/scientificreports) | (2020) 10:11323

2.Conference:

  • Gold nanoparticles-mediated detection of single-stranded nucleic acids with a protein nanopore platform, Tudor Luchian, 2020 NANOPORE ELECTROCHEMISTRY MEETING Oct. 09 – Oct. 14, 2020, Online

3. Scientific report:

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The “state of the art” equipment for implementing the objectives include: patch-clamp amplifiers (Molecular Devices), FluoroMax-4 spectrofluorimeter (Horiba Jobin Yvon), UV-VIS spectrophotometers (Thermo Scientific), Peltier temperature regulator (Harvard Apparatus), a low volume isothermal titration calorimeter (Malvern Analytical), refrigerated centrifuge (Hettich) and other biochemistry items (https://erris.gov.ro/MOLECULAR-BIOPHYSICS-AND-MED).

Finanțare prin/Finance by: PN III Planul Naţional de Cercetare, Dezvoltare şi Inovare  2015 – 2020, Programul 1: Dezvoltarea sistemului național de CD, Subprogramul 1.1 Resurse umane

TE: https://uefiscdi.gov.ro/proiecte-de-cercetare-pentru-stimularea-tinerelor-echipe-independente

Contract nr. TE 18/ 01.09.2020

COD: PN-III-P1-1.1-TE-2019-0037

Implementare /Implementation period: 01.09.2020- 31.08.2022

Valoare contract/Contract value: 431,900.00 lei

Director proiect/Project Manager: Conf. univ.dr. Loredana MEREUȚĂ

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Contact: e-mail: loredana.mereuta@uaic.ro