Detectia multiplex, cu sensibilitate si selectivitate moleculara, a unor miRNAs relevante fiziologic, cu ajutorul unor xeno acizi nucleici
Xeno nucleic acids-mediated, real-time multiplexed detection of disease relevant miRNAs, with single molecule sensitivity and selectivity
The overall aim of the project is to use a rationally designed library of particular xeno nucleic acids, namely peptide-nucleic acids (PNAs) conjugated with variable sized poly(Arg) tags (probe carriers), to serve – in conjunction with protein nanopores working as transducers – as multiplexed, real-times sensing probes for target, single-molecule miRNAs.
Abstract: miRNAs are small non-coding RNAs which regulate gene expression at post-translational level, and the content of circulating miRNAs in biological fluids changes significantly during the onset and progress of major diseases. Therefore, miRNAs became important biomarkers, that could be used for disease diagnosis and prognosis. Herein we address fundamental problems in miRNA detection, by proposing a paradigm to allow their multiplexed, real-time monitoring, offering highly accurate electronic readout of miRNA profiles, without the need of labeling or amplification, thus rendering the approach straightforward and efficient. Our innovative strategy is set on three pillars: (i) we will use a nanopore platform as a single-molecule transducer of miRNA detection, enabling real-time, sensitive and selective miRNA detection from similar sequences; (ii) to achieve enhanced selectivity detection for miRNAs, we will employ as sensing elements a particular class of xeno nucleic acids, peptide-nucleic acids (PNA), functionalized with various length polycationic tags; (iii) the tactics of the proposed approach will result in the sensitive, multiplexed detection of different miRNA species during the same experiment. With PNA-conjugated hybrid nanopores working as arrays and miniature amplifiers built on custom ASICs, we envision that the results stemming from this proposal will pave the way toward cheap, fast and accurate, simultaneous detection of multiple miRNAs in point-of-care units.
O1. Macroscopic characterization of PNA-miRNA base-paired constructs.
O2. Proof-of-concept of reproducible signature detection of poly(Arg)-functionalized PNAs, miRNAs and PNA- miRNA duplexes with the a-HL nanopore.
O3. The specificity of the a-HL nanopore-based assay in detecting miRNAs. Dielectrophoretic mechanism of poly(Arg)-functionalized PNA-miRNA capture by a single nanopore.
O4. Proof-of-concept for multiplexed profiling of distinct miRNAs in electrolyte buffers.
O5. Demonstrating the capability of the a-HL-based nano-sensor for the direct, multiplexed detection of miRNAs in biological samples.
Kinetic and steady-state characterization of poly(Arg)-PNAs-miRNA hybridization. Correlation of specific miRNAs presence with the capture time, relative blockade current, residual current and duration time of block. Quantitative descriptors of miRNA detection with probe PNAs, establishing the mismatch resolution, detection sensitivity, dynamic range of detection in various media. Real-time profiling of distinct target miRNAs from stochastic ionic current changes reflecting (probe)PNA-(target)miRNA duplexes-α-HL interactions. Sensitivity, reproducibility and selectivity assays testing of the miRNA detection procedure, via direct comparison with established protocols in the field.
Dissemination of scientific results
► Scientific articles
- Schiopu Irina, Asandei Alina, Mereuta Loredana, Dragomir Isabela, Bucataru Ioana Cezara, Luchian Tudor. Single-molecule detection and manipulation with biological nanopores. Studia Universitatis Babes-Bolyai, Chemia . 2021, 66 161-174
- Alina Asandei, Loredana Mereuta, Irina Schiopu, Yoonkyung Park, Tudor Luchian. Teaching an old dog new tricks: A lipid membrane-based electric immunosensor for real-time probing of the spike S-1 protein subunit from SARS-CoV-2. Proteomics, 2021, e2100047.
- Tudor Luchian, Loredana Mereuta, Yoonkyung Park, Alina Asandei, Irina Schiopu. Single-molecule, hybridization-based strategies for short nucleic acids detection and recognition with nanopores. Proteomics, 2021, e2100046.
- Isabela S Dragomir, Alina Asandei, Irina Schiopu, Ioana C Bucataru, Loredana Mereuta, Tudor Luchian. The Nanopore-Tweezing-Based, Targeted Detection of Nucleobases on Short Functionalized Peptide Nucleic Acid Sequences. Polymers, 2021, 13, 1210.
- Loredana Mereuta, Alina Asandei, Isabela Dragomir, Jonggwan Park, Yoonkyung Park, and Tudor Luchian. A Nanopore Sensor for Multiplexed Detection of Short Polynucleotides Based on Length-Variable, Poly-Arginine-Conjugated Peptide Nucleic Acids. Analytical Chemistry 2022, 94 (24), 8774-8782, DOI: 10.1021/acs.analchem.2c01587 ,Q1
- Alina Asandei, Loredana Mereuta, Ioana C. Bucataru, Yoonkyung Park, Tudor Luchian. A Single-Molecule Insight into the Ionic Strength-dependent, Cationic Peptide Nucleic Acids-Oligonucleotides Interactions. Chemistry An Asian Journal 2022, e202200261, DOI: 10.1002/asia.202200261, Q2
- 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, 12 (8), 596. Q1
- Sixth Edition of International Conference on Analytical and Nanoanalytical Methods for Biomedical and Environmental Sciences, „IC-ANMBES 2022”, June 8-10, 2022, Brasov, Romania
- Detection of nucleobases on short functionalized peptide-nucleic acid sequences using nanopore-tweezing method, Isabela S. Dragomir, Alina Asandei, Irina Schiopu, Ioana C. Bucataru, Loredana Mereuta, Tudor Luchian
- Protein nanopore-based method for sequence specific detection of single-stranded DNA using gold nanoparticles and peptide nucleic acids, Ioana Cezara Bucataru, Loredana Mereuta, Alina Asandei, Isabela Dragomir, Tudor Luchian
- A XVII-a Conferinţă Naţională de Biofizică, CNB 2022, 23-25 Septembrie 2022, Târgu Mureș, România
- A tug-of-war between electric forces: The nanopore-tweezing method applied in molecular sensing, Isabela S. Dragomir, Alina Asandei, Irina Schiopu, Ioana C. Bucataru, Loredana Mereuta, Tudor Luchian
► Scientific report
- Raport științific nr 1/2021 (.pdf – RO)/ Scientific report no 1/2021 (.pdf – EN)
- Raport științific nr. 2/2022 (.pdf-RO) / Scientific report no 2/2022 (.pdf -EN)
The project team:
- Prof. dr. Tudor LUCHIAN(Director proiect/Project leader)
- CS II dr. Alina ASANDEI
- Conf. dr. Loredana MEREUȚĂ
- CS III dr. Irina ȘCHIOPU
- ACS. dr. Isabela Dragomir
- PhD student Ioana Cezara Bucataru
Finanțare prin/Finance by: PN III Planul Naţional de Cercetare, Dezvoltare şi Inovare 2015 – 2020, Programul 4: Cercetare fundamentală și de frontieră
Contract nr. PCE 69 – 04.01.2021
Implementare/Implementation period: 04.01.2021- 31.12.2023
Valoare contract/ Contract value: 1.198.032,00 lei
Director proiect/Project Manager: Prof. dr. Tudor LUCHIAN
Contact: e-mail: email@example.com