Detection of low-frequency mutations in clinical samples by increasing mutation abundance via the excision of wild-type sequences.

The efficiency of DNA-enrichment techniques is often insufficient to detect mutations that occur at low frequencies. Here we report a DNA-excision method for the detection of low-frequency mutations in genomic DNA and in circulating cell-free DNA at single-nucleotide resolution. The method is based on a competitive DNA-binding-and-digestion mechanism, effected by deoxyribonuclease I (DNase) guided by single-stranded phosphorothioated DNA (sgDNase), for the removal of wild-type DNA strands. The sgDNase can be designed against any wild-type DNA sequences, allowing for the uniform enrichment of all the mutations within the target-binding region of single-stranded phosphorothioated DNA at mild-temperature conditions. Pretreatment with sgDNase enriches all mutant strands with initial frequencies down to 0.01% and leads to high discrimination factors for all types of single-nucleotide mismatch in multiple sequence contexts, as we show for the identification of low-abundance mutations in samples of blood or tissue from patients with cancer. The method can be coupled with next-generation sequencing, droplet digital polymerase chain reaction, Sanger sequencing, fluorescent-probe-based assays and other mutation-detection methods.

Dynamics of base pairs with low stability in RNA by solid-state nuclear magnetic resonance exchange spectroscopy.

Base pairs are fundamental building blocks of RNA. The base pairs of low stability are often critical in RNA functions. Here, we develop a solid-state NMR-based water-RNA exchange spectroscopy (WaterREXSY) to characterize RNA in solid. The approach uses different chemical exchange rates between iminos and water to evaluate base pair stability; the less stable ones would exchange more frequently, leading to stronger cross-peaks on WaterREXSY. Applied to the riboA71-adenine complex (the 71nt-aptamer domain of add adenine riboswitch from Vibrio vulnificus), the U47⋅U51 base pair, which is critical in ligand binding, was found to be less stable than other base pairs. The imino-water exchange rates of U47 at different temperatures are about 500-800 s-1, indeed indicative of low stability. This implies a highly complex and plastic triad involving U47⋅U51 and that the opening of the U47⋅U51 base pair may be the early stage of ligand release.

A multifunctional nanoprobe for targeting tumors and mitochondria with singlet oxygen generation and monitoring mitochondrion pH changes in cancer cells by ratiometric fluorescence imaging.

Mitochondria are the main sites of cell metabolism. Even minor pH changes may lead to mitochondrial dysfunction and promote cell apoptosis. Mitochondrion-targeting photosensitizers can produce singlet oxygen in the mitochondria. In tumor photodynamic therapy (PDT), tumor cells are killed through singlet oxygen generation by photosensitizers, and optimally the process of cell apoptosis can be real-time monitored by monitoring the changes of mitochondrial pH value. To this end, a multifunctional nanoprobe that is not only able to produce singlet oxygen in mitochondria but also able to detect the changes in mitochondrial pH value has been developed in this work. The probe is a single-excited dual-emission biomass quantum dot (BQD-FA) prepared from Osmanthus leaves with folic acid (FA) and polyoxyethylene diamine as modifiers. The BQD-FAs can target tumor cells and mitochondria, and produce singlet oxygen in the mitochondria under near-infrared laser irradiation (λ em = 660 nm). On the other hand, in the pH range of 3-8, the fluorescence intensity ratio of BQD-FAs at wavelengths 490 nm and 650 nm showed a good linear relationship with the pH value of mitochondria. The ratiometric fluorescence imaging of mitochondria using the prepared BQD-FAs showed that when the cells were chemically stimulated with chlorphenizone, the mitochondrial pH dropped from 7.9 to 7.2 within 15 min. Based on these characteristics, we envision that the prepared multifunctional nanoprobe will be of high significance in the biomedical research of mitochondria and PDT of tumors.

Unique Approach To Develop Carbon Dot-Based Nanohybrid Near-Infrared Ratiometric Fluorescent Sensor for the Detection of Mercury Ions.

The ratiometric fluorescence assay, which can eliminate the external effects, has attracted great attention. In this work, a carbon dot (CD)-based nanohybrid dual-emission system was simply prepared by a unique approach of solvothermal treating corn bract and used as a ratiometric fluorescent sensor for Hg2+ detection. Under a single excitation, the obtained nanohybrid sensor had two emission bands around 470 and 678 nm, which may originate from the intrinsic structure of CDs and chlorophyll-derived porphyrins, respectively. In the presence of Hg2+, the fluorescence at 678 nm could be remarkably quenched, while the fluorescence intensity at 470 nm was only slightly altered. The fluorescence intensity ratio at 470 and 678 nm exhibited a good linear relationship in the Hg2+ concentration range from 0 to 40 µM with a detection limit of about 9.0 nM. It also had a satisfying assay performance in serum and river water samples. The prepared CD-based nanohybrid sensor here may hold the further potential applications in biomedicine study, environmental protection, and food safety.

Sulfur and nitrogen binary doped carbon dots derived from ammonium thiocyanate for selective probing doxycycline in living cells and multicolor cell imaging.

A novel sulfur and nitrogen binary doped carbon dots (S,N-CDs) was synthesized by one-step manner through the hydrothermal treatment of citric acid (CA) and ammonium thiocyanate, and the procedures for biomedical applications, including probing doxycycline in living cells and multicolor cell imaging were developed. The obtained S,N-CDs are stable in aqueous solution, possess a very high quantum yield (QY, 74.15%) and good photostability. The fluorescence of S,N-CDs can be specifically quenched by doxycycline, providing a convenient turn-off assay of doxycycline. This assay shows a wide linear detection range from 0.08 to 60 µM with a low detection limit of 20 nM. The present method also displays a good selectivity. More importantly, the S,N-CDs have an excellent biocompatibility and low cytotoxicity, allowing the multicolor cell imaging and doxycycline detection in living cells. Consequently, the developed doxycycline methods is facile, low-cost, biocompatible, sensitive and selective, which may hold the potential applications in the fields of food safety and environmental monitoring, as well as cancer therapy and related mechanism research.

Green preparation of fluorescent carbon dots from lychee seeds and their application for the selective detection of methylene blue and imaging in living cells.

A green approach was developed for the preparation of fluorescent carbon dots (CDs) by using lychee seeds as precursors. The preparation of CDs was performed by simple pyrolysis. The quantum yield of as-prepared CDs was 10.6% by using quinine sulfate as the reference. The CDs were employed as fluorescence probes for the detection of methylene blue (MB). This sensing system exhibits excellent sensitivity and selectivity toward MB, and a detection limit of 50 mM is achieved. The possible application of as-prepared CDs for imaging in living cells was also explored. The inherent cytotoxicity of CDs was evaluated using HepG2 cell, and the cell viabilities were estimated to be greater than 90% upon addition of the CDs over a wide concentration range of 0-1000 µg mL-1. It was then successfully applied for the fluorescence imaging of HepG2 cells.