Abstract
Short single-stranded nucleic acids as found in a variety of bodily fluids have recently emerged as minimally invasive biomarkers for a broad range of pathologies, most notably cancer. Because of their small size, low natural abundance and high sequence homology between family members they are challenging to detect using standard technologies suitable for use at the point-of-care. Herein we report the design, engineering and testing of a novel sensing strategy: electrochemically active molecular probes based on peptide nucleic acid (PNA) scaffolds for the detection of single-stranded oligonucleotides, in particular microRNAs (or miRs). As a proof-of-principle, a wide range of probes were designed and tested to detect miR-141, a known diagnostic biomarker for prostate cancer. Optimal quantitative sensing of miR-141 was achieved via the first example of an electrochemical oligonucleotide-templated reaction (EOTR), whereby two PNA probes - functionalized with an aniline and a 1,4-catechol respectively - preferentially react with each other upon simultaneous hybridization to the same RNA target strand, serving here as a template. Quantitative, electrochemical detection of the product of this bio-orthogonal reaction showed direct correlation between adduct formation and miR-141 concentration. Coupling the specificity of OTR with the speed and sensitivity of electrochemical sensing delivers EOTRs as a promising new technique for fast, low-cost, quantitative and sequence-specific detection of short nucleic acids from liquid biopsies.
| Original language | English |
|---|---|
| Article number | 112891 |
| Journal | Biosensors and Bioelectronics |
| Volume | 176 |
| Early online date | 15 Dec 2020 |
| DOIs | |
| Publication status | Published - 15 Mar 2021 |
Bibliographical note
Funding Information:Two PNAs complementary to miR-141 were synthesized in-house via solid phase peptide synthesis following a previously described procedure (Metcalf et al., 2016). In short, 7-mer PNA probes functionalized with an aniline (PNA-A) or catechol (PNA-C) moiety were synthesized on solid support using chloro-trityl polystyrene resin and preloaded Fmoc-Arg (Pbf)-Wang resin (Merck Biosciences) (Table 1), respectively. Aniline was introduced at the C-terminus of PNA-A by reacting chloro-trityl resin with 1,4-phenylenediamine (8 mol equiv.) and N,N-diisopropylethylamine (DIPEA) (8 mol equiv) in DMF as previously reported (Abbenante et al., 2000). 1,4-catechol was introduced at the N-terminus of PNA-C by coupling 3,4-dihydroxyphenylacetic acid (DOPAC) onto the PNA's terminal free amine. To ensure sufficient water solubility at physiological pH, two arginine residues were also introduced at the C- and N- terminus of PNA-C and PNA-A, respectively. Functionalized PNAs were cleaved from solid support by treatment with a solution of trifluoroacetic acid (TFA): triisopropylsilane (TIPS): de-ionised water (resistivity >18 MΩ cm) (95:2.5:2.5, v/v). The desired PNAs were purified using high-performance liquid chromatography (HPLC) and characterized by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) using sinapinic acid as a matrix (Figure S1, supplementary information).This work was supported by PhD scholarships from BBSRC (P.G.) and from China Scholarship Council (X.M.) and by a research grant from Cancer Research UK (N.I. and R.C. C49996/A26141).
Funding Information:
This work was supported by PhD scholarships from BBSRC (P.G.) and from China Scholarship Council (X.M.) and by a research grant from Cancer Research UK (N.I. and R.C., C49996/A26141 ).
Publisher Copyright:
© 2020 Elsevier B.V.