A mutation-sensitive, multiplexed and amplification-free detection of nucleic acids by stretching single-molecule tandem hairpin probes.

The detection of nucleic acid sequences in parallel with the discrimination of single nucleotide variations (SNVs) is critical for research and clinical applications. A few limitations make the detection technically challenging, such as too small variation in probe-hybridization energy caused by SNVs, the non-specific amplification of false nucleic acid fragments and the few options of dyes limited by spectral overlaps. To circumvent these limitations, we developed a single-molecule nucleic acid detection assay without amplification or fluorescence termed THREF (hybridization-induced tandem DNA hairpin refolding failure) based on multiplexed magnetic tweezers. THREF can detect DNA and RNA sequences at femtomolar concentrations within 30 min, monitor multiple probes in parallel, quantify the expression level of miR-122 in patient tissues, discriminate SNVs including the hard-to-detect G-U or T-G wobble mutations and reuse the probes to save the cost. In our demonstrative detections using mock clinic samples, we profiled the let-7 family microRNAs in serum and genotyped SARS-CoV-2 strains in saliva. Overall, the THREF assay can discriminate SNVs with the advantages of high sensitivity, ultra-specificity, multiplexing, reusability, sample hands-free and robustness.

ZYG11B potentiates the antiviral innate immune response by enhancing cGAS-DNA binding and condensation.

As a key dsDNA recognition receptor, cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS) plays a vital role in innate immune responses. Activated cGAS, by sensing DNA, catalyzes the synthesis of the secondary messenger cyclic GMP-AMP (cGAMP), which subsequently activates downstream signaling to induce production of interferons and inflammatory cytokines. Here, we report Zyg-11 family member B (ZYG11B) as a potent amplifier in cGAS-mediated immune responses. Knockdown of ZYG11B impairs production of cGAMP and subsequent transcription of interferon and inflammatory cytokines. Mechanistically, ZYG11B enhances cGAS-DNA binding affinity, potentiates cGAS-DNA condensation, and stabilizes the cGAS-DNA condensed complex. Moreover, herpes simplex virus 1 (HSV-1) infection induces ZYG11B degradation in a cGAS-unrelated manner. Our findings not only reveal an important role of ZYG11B in the early stage of DNA-induced cGAS activation but also indicate a viral strategy to dampen the innate immune response.

Cytosine Methylation Enhances DNA Condensation Revealed by Equilibrium Measurements Using Magnetic Tweezers.

CpG methylation of DNA is common in mammalian cells. In sperm, the DNA has the highest level of CpG methylation and is condensed into toroidal structures. How CpG methylation affects DNA structures and interactions is important to understand its biological roles but is largely unknown. Using an RNA-DNA-RNA structure, we observed the equilibrium hopping dynamics between the condensed and extended states of DNA in the presence of polyamines or polylysine peptide as a reduced model of histone tails. Combing with the measured DNA elasticities, we report that CpG methylation of each cytosine nucleotide substantially increases DNA-DNA attraction by up to 0.2 kBT. For the DNA with 57% GC content, the relative increase caused by CpG methylation is up to 32% for the spermine-induced DNA-DNA attraction and up to 9% for the polylysine-induced DNA-DNA attraction. These findings help us to evaluate the energetic contributions of CpG methylation in sperm development and chromatin regulation.

A Universal Assay for Making DNA, RNA, and RNA-DNA Hybrid Configurations for Single-Molecule Manipulation in Two or Three Steps without Ligation.

Despite having a great variety of topologies, most DNA, RNA, and RNA-DNA hybrid (RDH) configurations for single-molecule manipulation are composed of several single-stranded (ss) DNA and ssRNA strands, with functional labels at the two ends for surface tethering. On this basis, we developed a simple, robust, and universal amplification-annealing (AA) assay for making all these configurations in two or three steps without inefficient digestion and ligation reactions. As examples, we made ssDNA, short ssDNA with double-stranded (ds) DNA handles, dsDNA with ssDNA handles, replication-fork shaped DNA/RDH/RNA, DNA holiday junction, three-site multiple-labeled and nicked DNA, torsion-constrained RNA/RDH, and short ssRNA with RDH handles. In addition to single-molecule manipulation techniques including optical tweezers, magnetic tweezers, and atomic force microscopy, these configurations can be applied in other surface-tethering techniques as well.

S-DNA and RecA/RAD51-Mediated Strand Exchange in Vitro.

S-DNA (stretched DNA) is an elongated base-paired DNA conformation under high tension. Because the RecA/Rad51 family DNA recombinases form helical filaments on DNA and mediate the formation of the DNA triplex (D-loop), in which the DNA is stretched, and because the extension of these nucleoprotein filaments is similar to the extension of S-DNA, S-DNA has long been hypothesized as a possible state of DNA that participants in RecA/Rad51-mediated DNA strand exchange in homologous recombination. Such a hypothesis, however, is still lacking direct experimental studies. In this work, we have studied the polymerization and strand exchange on S-DNA mediated by Escherichia coli RecA, human Rad51, and Saccharomyces cerevisiae Rad51 by single-molecule magnetic tweezers. We report that RecA/Rad51 polymerizes faster on S-DNA than on B-DNA with the same buffer conditions. Furthermore, the RecA/Rad51-mediated DNA triplex forms faster from S-DNA than from B-DNA together with the homologous single-stranded DNA. These results provide evidence that S-DNA can interact with RecA and Rad51 and shed light on the possible functions of S-DNA.