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

-RNANANODETECT-

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.

Project objectives:

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.

Estimated results:

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.

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Dissemination of scientific results


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

PCE: https://uefiscdi.gov.ro/proiecte-de-cercetare-exploratorie

Contract nr. PCE 69 – 04.01.2021

COD: PN-III-P4-ID-PCE-2020-0011

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

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Contact: e-mail: luchian@uaic.ro