Quantification of plasma HIV RNA using chemically engineered peptide nucleic acids

Abstract

The remarkable stability of peptide nucleic acids (PNAs) toward enzymatic degradation makes this class of molecules ideal to develop as part of a diagnostic device. Here we report the development of chemically-engineered PNAs for the quantitative detection of HIV RNA at clinically relevant levels that are competitive with current PCR-based assays. Using a sandwich hybridization approach, chemical groups were systematically introduced into a surface PNA probe and a reporter PNA probe to achieve quantitative detection for HIV RNA as low as 20 copies per milliliter of plasma. For the surface PNA probe, four cyclopentane groups were incorporated to promote stronger binding to the target HIV RNA compared to PNA without the cyclopentanes. For the reporter PNA probe, 25 biotin groups were attached to promote strong signal amplification after binding to the target HIV RNA. These general approaches to engineer PNA probes may be used to detect other RNA target sequences.

A PNA‐DNA2 Triple‐Helix Molecular Switch‐Based Colorimetric Sensor for Sensitive and Specific Detection of microRNAs from Cancer Cells

“…The homopurine PNA oligonucleotide was manually prepared by the method of solidphase peptide synthesis and characterized according to our previously published literature. [49,50] All the PNA oligomer was purified by high-quality pre-packed column (Agilent Eclipse XDB-C18) and characterized by mass analysis. Their purities were greater than 99 % according to HPLC (Agilent).…”

Highly sensitive and specific screening of EGFR mutation using a PNA microarray-based fluorometric assay based on rolling circle amplification and graphene oxide

“…PNA oligomers were synthesized on 10 mmol scale using Fmocsolid phase peptide synthesis protocols on MBHA resin with HBTU as the amide-forming reagent. [56][57][58] The Fmoc as protecting group was cleaved by 20 mL 20% piperidine DMF solution. And the uncoupling amino groups were capped using 2 mL acetic anhydride/pyridine/NMP (1 : 2 : 2, v/v).…”

“…A 11-nt PNA capture probe, which is used to recognize and capture target EGFR sequence, was designed and synthesized by the method of solid-phase peptide synthesis as described previously. [56][57][58] To increase PNA solubility and make it suitable for surface anchoring, terminal amino modied mPEG linker (NH 2 -(mPEG) 3 -) was also attached to the probe. In this study, the rst step is to prepare PNA microarrays by attaching PNA capture probe covalently via amide bond to the plastic surface of a NUNC 96-well plate.…”

Application of nanotechnology in biosensors for enhancing pathogen detection

“…RNA has also been used for biosensor probes (Ferapontova, Olsen, & Gothelf, ; Ke et al, ), but is generally less stable than DNA based probes because of its susceptibility to ribonuclease digestion and chemical cleavage (Ferapontova et al, ). More recently, PNA, a synthetic analog to DNA/RNA with a backbone composed of repeating N ‐(2‐aminoethyl)‐glycine units linked by amide bonds (Nielsen & Egholm, ), has also been employed for pathogen detection (Kuhn et al, ; Mateo‐Marti, Briones, Pradier, & Martin‐Gago, ; Metaferia et al, ; Ozkan et al, ; Steichen, Decrem, Godfroid, & Buess‐Herman, ; X. Su, Teh, Lieu, & Gao, ; Uno, Tabata, & Kawai, ; J. Wang et al, ; C. Zhao et al, ). PNA recognition layers impart remarkable sequence specificity and offers greater flexibility in reaction conditions over DNA/RNA based probes (Brandt & Hoheisel, ; J. Wang et al, ).…”