Novel Signal-Enhancing Approaches for Optical Detection of Nucleic Acids—Going beyond Target Amplification

Abstract

Detection of low-abundance nucleic acids is a challenging task, which over the last two decades has been solved using enzymatic target amplification. Enzymatic synthesis enhances the signal so that diverse, scientifically and clinically relevant molecules can be identified and studied, including cancer DNA, viral nucleic acids, and regulatory RNAs. However, using enzymes increases the detection time and cost, not to mention the high risk of mistakes with amplification and data alignment. These limitations have stimulated a growing interest in enzyme-free methods within researchers and industry. In this review we discuss recent advances in signal-enhancing approaches aimed at nucleic acid diagnostics that do not require target amplification. Regardless of enzyme usage, signal enhancement is crucial for the reliable detection of nucleic acids at low concentrations. We pay special attention to novel nanomaterials, fluorescence microscopy, and technical advances in detectors for optical assessment. We summarize sensitivity parameters of the currently available assays and devices which makes this review relevant to the broad spectrum of researchers working in fields from biophysics, to engineering, to synthetic biology and bioorganic chemistry.

Revealing Nucleic Acid Mutations Using Förster Resonance Energy Transfer-Based Probes

“…Interactions of FRET probes with nucleic acids become even more complicated when the detection is to be performed directly in cells. Unlike short oligonucleotides, intracellular DNA and RNA targets are long biopolymers which are present in complex intracellular matrices at extremely low concentrations, often only a couple of molecules per cell [ 1 , 2 ]. For the last three decades, low abundance of nucleic acids has been approached using enzymatic amplification including polymerase chain reaction (PCR) [ 28 ].…”

“…This requires photostability of both the acceptor and the donor, since bleaching in one or both fluorophores might lead to non-stoichiometric conditions [ 11 , 12 , 15 , 16 , 17 , 18 ]. Nowadays, many dyes suffer from a low quantum yield and brightness [ 2 ]. This can be improved using bright fluorescent dyes and materials.…”

“…This can be improved using bright fluorescent dyes and materials. Among others, it can be lanthanide chelates, fluorescently labelled organic polymers such as oligo ethylene glycol methacrylate (OEGMA) nanoparticles, and QDs [ 2 ]. Noteworthy, optical properties crucially depend on the purity of the aforementioned molecules [ 16 ].…”

“…To ensure these functions, nucleic acids actively interact with each other and other biomolecules, change conformations and move within living cells. So far, obtaining data on nucleic acids in a biologically relevant context has been hindered by the lack of ultra-sensitive methods and reliable probes [ 2 ]. The recent progress in biophysics and chemistry of nucleic acids has provided an opportunity to open up the black box of nucleic acid structure and interactions directly in cells and to quantify nucleic acids in diverse biological environments [ 2 ].…”

“…This theoretical framework is confirmed by extensive experimental data in vitro, at concentrations above 50 nM and for unmodified and modified sequences [ 5 ]. A forthcoming challenge for the theory and design of oligonucleotide probes is that natural DNA and RNA are long biopolymers which are present in complex intracellular matrices at extremely low concentrations, often only a couple of molecules per cell [ 1 , 2 ].…”