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.

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.

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.]

Dissemination of scientific results:

 1.Article: 

• Mereuta, L; Asandei, A; Dragomir, IS; Bucataru, IC; Park, J; Seo, CH; Park, Y;  Luchian, T. Sequence-specific detection of single-stranded DNA with a gold nanoparticle-protein nanopore approach ; Scientific Reports (2020), 10:11323
• Asandei, A; Mereuta, L; Schiopu, I; Park, Y; Luchian, T. Teaching an old dog new tricks: A lipid membranebased electric immunosensor for real-time probing of the spike S-1 protein subunit from SARS-CoV-2;  PROTEOMICS (2021), DOI10.1002/pmic.202100047
• Luchian, T; Mereuta, L; Park, Y; Asandei, A; Schiopu, I. Single-molecule, hybridization-based strategies for short nucleic acids detection and recognition with nanopores; PROTEOMICS (2021), DOI10.1002/pmic.202100046
• Dragomir, IS; Asandei, A; Schiopu, I; Bucataru, IC; Mereuta, L; Luchian, T. The Nanopore-Tweezing-Based, Targeted Detection of Nucleobases on Short Functionalized Peptide Nucleic Acid Sequences; POLYMERS (2021), DOI10.3390/polym13081210
• Schiopu, I; Asandei, A; Mereuta, L; Dragomir, I; Bucataru, C; Luchian, T. Single-Molecule Detection and Manipulation with Biological Nanopores; STUDIA UNIVERSITATIS BABES-BOLYAI CHEMIA (2021), DOI10.24193/subbchem.2021.3.09
• Asandei, Alina; Mereuta, Loredana; Bucataru, Ioana C.; Park, Yoonkyung; Luchian, Tudor; A Single-Molecule Insight into the Ionic Strength-dependent, Cationic Peptide Nucleic Acids-Oligonucleotides Interactions , CHEMISTRY-AN ASIAN JOURNAL (2022)
• Mereuta, Loredana; Asandei, Alina; Dragomir, Isabela; Park, Jonggwan; Park ,Yoonkyung; Luchian, Tudor; A Nanopore Sensor for Multiplexed Detection of Short Polynucleotides Based on Length-Variable, Poly-Arginine- Conjugated Peptide Nucleic Acids, Analytical Chemistry (2022)
• Ioana C., Bucataru; Isabela, Dragomir; Alina, Asandei; Ana-Maria, Pantazica; Alina, Ghionescu; Norica, Branza-Nichita; Yoonkyung, Park; Tudor, Luchian;Probing the Hepatitis B Virus E-Antigen with a Nanopore Sensor Based on Collisional Events Analysis , Biosensors (2022)

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
• Detection of nucleobases on short functionalized peptide-nucleic acid sequences using nanopore-tweezing method, 2022 International Conference on Analytical and Nanoanalytical Methods for Biomedical and Environmental Sciences, Poster
• Protein nanopore-based method for sequence specific detection of single-stranded DNA using gold nanoparticles and peptide nucleic acids, 2022 International Conference on Analytical and Nanoanalytical Methods for Biomedical and Environmental Sciences, Poster

3. Scientific report:

• Scientific report no. 1/ 2020 (.pdf-RO)
• Scientific report no.2/2021 (.pdf-RO)
• Scientific report no.3/2022 (.pdf-RO)

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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).