Nucleic acid fluorescent probes are playing increasingly important roles in biological sensing in recent years. In addition to the conventional functions of single-stranded DNA/RNA to hybridize with their complementary strands, affinity nucleic acids (aptamers) with specific target binding properties have also been developed, which has greatly broadened the application of nucleic acid fluorescent probes to the detection of a large variety of analytes, including small molecules, proteins, ions, and even whole cells. Another chemical property of nucleic acids is to act as substrates for various nucleic acid enzymes. This property can be utilized not only to detect those enzymes and screen their inhibitors, but also employed to develop effective signal amplification systems, which implies extensive applications. This review mainly covers the biosensing methods based on the above three types of nucleic acid fluorescent probes. The most widely used intensity-based biosensing assays are covered first, including nucleic acid probe-based signal amplification methods. Then fluorescence lifetime, fluorescence anisotropy, and fluorescence correlation spectroscopy assays are introduced, respectively. As a rapidly developing field, fluorescence imaging approaches are also briefly summarized.

Enzymes containing 3'-5' exonuclease activities play vital roles in maintaining genome stability. Though a wide variety of methods have been developed for detection of these enzymes, few of them can be directly applied for in situ and real-time monitoring of the secretion of these active substances by living cells. Taking advantages of the free 3'-end of stacked guanine-quenched photoinduced electron transfer fluorescent probes, here we demonstrate a novel assay capable of in situ and real-time monitoring of the 3'-5' exonucleases secreted by living cells. The detection limit of the new method achieved as low as 0.04 U/mL, allowing direct monitoring of the target enzymes in an extracellular environment without preconcentration steps. False positive signals caused by other nonspecific enzymes were easily ruled out by the use of a control probe with the 3'-end modified with exonuclease-resistant phosphorothioate guanines. Using Alexa Fluor 488 as the fluorophore, the probe is adaptable to a wide range of pH conditions. The approach was successfully applied for in situ, real-time monitoring of the 3'-5' exonucleases secreted by suspension cells of Arabidopsis thaliana. It also holds great potential for in situ and real-time detection of many other DNA end-processing enzymes produced by other types of cells.

Lactic acid (LA) metabolism in the tumor microenvironment contributes to the establishment and maintenance of immune tolerance. This pathway is characterized in tumor associated macrophages. However, the role and pathway of LA metabolism at maternal-fetal interface during early pregnancy, especially in decidual macrophage differentiation, are still unclear. Herein, for the first time, we discovered that LA can trigger either M2 or M1 macrophage polarization via oxidative phosphorylation and glycolysis regulation under normoxia or hypoxia, respectively. Also, LA metabolism played a vital role in decidual macrophages-mediated recurrent pregnancy loss (RPL), through HIF-1α/SRC/LDHA pathway. Moreover, blockade of LA intake with AZD3965 (MCT-1 inhibitor) could rescue pregnancy in an abortion-prone mouse model, suggesting a potential therapeutic target in RPL. Collectively, the present study identifies the previously unknown functions of LA metabolism in the differentiation of decidual macrophages in early normal pregnancy and RPL, and provides a potential therapeutic strategy in RPL by manipulating decidual macrophages' functions through LA metabolic pathway.

We present a novel strategy for the highly selective detection of single-nucleotide variation at room temperature, based on an extremely specific interaction between Lambda exonuclease (λ exo) and a chemically modified DNA structure.

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A novel signal amplification system that is applicable to any DNA sequence of interest has been developed by using a combination of apurinic/apyrimidinic probe and endonuclease IV. The system allows rapid, highly selective and sensitive detection of target DNA sequences at very low concentrations (10 fmol) or low abundance levels (1%).

Lambda exonuclease (λ exo) plays an important role in the resection of DNA ends for DNA repair. Currently, it is also a widely used enzymatic tool in genetic engineering, DNA-binding protein mapping, nanopore sequencing and biosensing. Herein, we disclose two noncanonical properties of this enzyme and suggest a previously undescribed hydrophobic interaction model between λ exo and DNA substrates. We demonstrate that the length of the free portion of the substrate strand in the dsDNA plays an essential role in the initiation of digestion reactions by λ exo. A dsDNA with a 5′ non-phosphorylated, two-nucleotide-protruding end can be digested by λ exo with very high efficiency. Moreover, we show that when a conjugated structure is covalently attached to an internal base of the dsDNA, the presence of a single mismatched base pair at the 5′ side of the modified base may significantly accelerate the process of digestion by λ exo. A detailed comparison study revealed additional π–π stacking interactions between the attached label and the amino acid residues of the enzyme. These new findings not only broaden our knowledge of the enzyme but will also be very useful for research on DNA repair and in vitro processing of nucleic acids.

Sensitive and specific DNA hybridization is essential for nucleic acid chemistry. Competitive composition of probe and blocker has been the most adopted probe design for its relatively high sensitivity and specificity. However, the sensitivity and specificity were inversely correlated over the length and concentration of the blocker strand, making the optimization process cumbersome. Herein, we construct a theoretical model for competitive DNA hybridization, which disclose that both the thermodynamics and kinetics contribute to the inverse correlation. Guided by this, we invent the 4-way Strand Exchange LEd Competitive DNA Testing (SELECT) system, which breaks up the inverse correlation. Using SELECT, we identified 16 hot-pot mutations in human genome under uniform conditions, without optimization at all. The specificities were all above 140. As a demonstration of the clinical practicability, we develop probe systems that detect mutations in human genomic DNA extracted from ovarian cancer patients with a detection limit of 0.1%.