Telomere G-triplex lights up Thioflavin T for RNA detection: new wine in an old bottle.

Few reports are found working on the features and functions of the human telomere G-triplex (ht-G3) though the telomere G-quadruplex has been intensely studied and widely implemented to develop various biosensors. We herein report that ht-G3 lights up Thioflavin T (ThT) and establish a sensitive biosensing platform for RNA detection by introducing a target recycling strategy. An optimal condition was selected out for ht-G3 to promote ThT to generate a strong fluorescence. Accordingly, an ht-G3-based molecular beacon was successfully designed against the corresponding RNA sequence of the SARS-CoV-2 N-gene. The sensitivity for the non-amplified RNA target achieves 0.01 nM, improved 100 times over the conventional ThT-based method. We believe this ht-G3/ThT-based label-free strategy could be widely applied for RNA detection.

Detection of SARS-CoV-2 RNA through tandem isothermal gene amplification without reverse transcription.

Diagnosis of SARS-CoV-2 infection through rapid, accurate, and sensitive testing is the most important and fundamental step in coping with the COVID-19 epidemic. We have developed a sensitive fluorometric assay to detect SARS-CoV-2 viral RNA without thermal cycling. This assay system, based on tandem isothermal gene amplification (TIGA), is composed of ternary rolling circle amplification (t-RCA) and subsequent strand displacement amplification (SDA) coupled with G-quadruplex-generating RCA (SDA/GQ-RCA). Without the need to convert viral RNA into cDNA, viral RNA forms a ternary complex composed of hairpin primer (HP) and dumbbell padlock DNA during the t-RCA process. t-RCA generates a long chain of single-stranded DNA (ssDNA) with tandemly repeated hairpin structures that are subjected to SDA. SDA produces multiple short ssDNAs from t-RCA products, which then serve as primers for the second RCA reaction. A long ssDNA harboring repeated copies of the G-quadruplex is produced in the second round of RCA. Emission of enhanced fluorescence by thioflavin T, which intercalates into the G-quadruplex, allows fluorometric detection of amplified viral genes. This fluorometric analysis sensitively detected SARS-CoV-2 RNA as low as 5.9 aM, with a linear range between 0.2 fM and 200 fM within 1 h. Hence, this isothermal gene amplification method without reverse transcription of viral RNA can be applied to diagnose COVID-19 with high sensitivity and accuracy as an alternative to current PCR-based diagnosis.

Amyloid and Hydrogel Formation of a Peptide Sequence from a Coronavirus Spike Protein

We demonstrate that a conserved coronavirus spike protein peptide forms amyloid structures, differing from the native helical conformation and not predicted by amyloid aggregation algorithms. We investigate the conformation and aggregation of peptide RSAIEDLLFDKV, which is a sequence common to many animal and human coronavirus spike proteins. This sequence is part of a native alpha-helical S2 glycoprotein domain, close to and partly spanning the fusion sequence. This peptide aggregates into beta-sheet amyloid nanotape structures close to the calculated pI = 4.2, but forms disordered monomers at high and low pH. The beta-sheet conformation revealed by FTIR and circular dichroism (CD) spectroscopy leads to peptide nanotape structures, imaged using transmission electron microscopy (TEM) and probed by small-angle X-ray scattering (SAXS). The nanotapes comprise arginine-coated bilayers. A Congo red dye UV-vis assay is used to probe the aggregation of the peptide into amyloid structures, which enabled the determination of a critical aggregation concentration (CAC). This peptide also forms hydrogels under precisely defined conditions of pH and concentration, the rheological properties of which were probed. The observation of amyloid formation by a coronavirus spike has relevance to the stability of the spike protein conformation (or its destabilization via pH change), and the peptide may have potential utility as a functional material. Hydrogels formed by coronavirus peptides may also be of future interest in the development of slow-release systems, among other applications.

Unified-amplifier based primer exchange reaction (UniAmPER) enabled detection of SARS-CoV-2 from clinical samples.

Primer exchange reaction (PER) is an emergent method for non-templated synthesis of single stranded DNA molecules. PER has been shown to be effective in cell imaging systems and for detection of macromolecules. A particular application of PER is to detect a specific target nucleic acid. To this endeavor, two coupled DNA hairpins, a detector and an amplifier, play in accordance to extend a target nucleic acid with a concatemer DNA sequence. Here we introduced unified-amplifier based primer exchange reaction (UniAmPER) that beneficially extends the target by a unified-amplifier. The unified-amplifier operates as both detector and amplifier hairpins. The extension resulted in synthesis of concatemer G-rich sequences. The G-rich sequences were expected to form G-quadruplex (GQ) structures. Presence of the GQ structures were investigated by peroxidase activity of GQs in presence of hemin, H2°2 and 3,3',5,5'-Tetramethylbenzidine (TMB) as well as by fluorescence signal generation upon intercalation of thioflavin T (ThT). The presented unified-amplifier in this study facilitates application of PER systems for development of colorimetric or fluorogenic biosensors. As a proof of principle, the method has been applied for detection of reversely transcribed cDNAs from clinical SARS-CoV-2 samples.