A versatile and convenient tool for regulation of DNA strand displacement and post-modification on pre-fabricated DNA nanodevices.

Toehold-mediated strand displacement and its regulatory tools are fundamental for DNA nanotechnology. However, current regulatory tools all need to change the original sequence of reactants, making the regulation inconvenient and cumbersome. More importantly, the booming development of DNA nanotechnology will soon promote the production of packaged and batched devices or circuits with specified functions. Regarding standardized, packaged DNA nanodevices, access to personalized post-modification will greatly help users, whereas none of the current regulatory tools can provide such access, which has greatly constrained DNA nanodevices from becoming more powerful and practical. Herein, we developed a novel regulation tool named Cap which has two basic functions of subtle regulation of the reaction rate and erasability. Based on these functions, we further developed three advanced functions. Through integration of all functions of Cap and its distinct advantage of working independently, we finally realized personalized tailor-made post-modification on pre-fabricated DNA circuits. A pre-fabricated dual-output DNA circuit was successfully transformed into an equal-output circuit, a signal-antagonist circuit and a covariant circuit according to our requirements. Taken together, Cap is easy to design and generalizable for all strand displacement-based DNA nanodevices. We believe the Cap tool will be widely used in regulating reaction networks and personalized tailor-made post-modification of DNA nanodevices.

Guiding-Strand-Controlled DNA Nucleases with Enhanced Specificity and Tunable Kinetics for DNA Mutation Detection.

Nucleases are powerful tools in various biomedical applications, such as genetic engineering, biosensing, and molecular diagnosis. However, the commonly used nucleases (endonuclease IV, apurinic/apyrimidinic endonuclease-1, and λ exonuclease) are prone to the nonspecific cleavage of single-stranded DNA, making the desired reactions extremely low-yield and unpredictable. Herein, we have developed guiding-strand-controlled nuclease systems and constructed theoretical kinetic models to explain their mechanisms of action. The models displayed excellent agreement with the experimental results, making the kinetics highly predictable and tunable. Our method inhibited the nonspecific cleavage of single-stranded probes while maintaining highly efficient cleavage of double-stranded DNA. We also demonstrated the clinical practicability of the method by detecting a low-frequency mutation in a genomic DNA sample extracted from the blood of a patient with cancer. The limit of detection could be 0.01% for PTEN rs121909219. We believe that our findings provide a powerful tool for the field and the established model provides us a deeper understanding of the enzymatic activities of DNA nucleases.

Self-Internal-Reference Probe System for Control-Free Quantification of Mutation Abundance.

Gene mutations are important biomarkers for the diagnosis, classification, monitoring, and prognosis evaluation of cancers and genetic diseases. Both personalized cancer treatment and noninvasive prenatal testing require methods to accurately determine the abundance of mutation. At present, the widely adopted and convenient methods for measuring mutation abundance are mainly based on relative quantification, which requires negative samples and strict control of the analyte amounts. The development of DNA-probe-based methods that can determine the mutation abundance without negative samples nor control of analyte amount is highly preferred. The key to solving this bottleneck lies in whether the probe's response to mutation abundance can be completely independent of the number of targeted DNA strands. Herein, we propose the design of a self-internal-reference probe system. We established a theoretical model of this system and used the model to guide the design of probes. In this model, we provided quantitative corrections to the test results from the internal reference, thereby eliminating the influence of substrate amount. Therefore, the purification and quantification processes toward polymerase chain reaction (PCR) amplicons can be omitted. We applied this system to analyze unquantified PCR products aimed at cancer mutation detection and noninvasive prenatal testing.

Branch migration-based polymerase chain reaction combined with endonuclease IV-assisted target recycling probe/blocker system for detection of low-abundance point mutations.

Sensitive detection of low-abundance point mutations in blood or tissue may provide a great opportunity for the minimally invasive diagnosis of cancer and other related diseases. We demonstrate a novel method for ultra-sensitive detection of point mutations at low abundance by combination of branch migration-based PCR with endonuclease IV-assisted target recycling probe/blocker system. The method is able to identify the point mutations at abundances down to 0.01-0.02%. We anticipate this method to be widely adopted in clinical diagnosis and molecular research.

Enhancing APE1 detection through apurinic/apyrimidinic site inhibition of DNA polymerase: an innovative, highly sensitive approach.

This study presents an innovative method for the highly sensitive detection of apurinic/apyrimidinic endonuclease 1 (APE1), a crucial biomarker and target for cancer diagnosis and treatment. The method is predicated on our discovery that the apurinic or apyrimidinic site (AP site) can inhibit the activity of Taq DNA polymerase. Subsequent experiments further led to the development of a new amplification method based on the digestion activity of Lambda exonuclease. This approach showed potential to detect trace amounts of APE1 in biological samples with high sensitivity.

Potential utility of ADNP in circulating tumor cells as biomarker for prognostics in non-muscle-invasive bladder cancer.

This study aims to evaluate the prognostic utility of Activity-dependent neuroprotective protein (ADNP) expression in Circulating Tumor Cells (CTCs) inpatients with Non-muscle-invasive Bladder Cancer (NMIBC) undergoing Transurethral Resection of Bladder Tumor (TURBT). A prospective cohort of 74 bladder cancer patients and 22 non-cancer controls were enrolled. The expression of ADNP mRNA was detected by immunomagnetic beads-droplet digital PCR. The ADNP mRNA expression was evaluated in patients with high-risk NMIBC and those with indeterminate invasion depth post 2nd TURBT. Primary cultured bladder cancer cells and PBMCs from healthy donors were immunofluorescence stained. Our findings suggest that baseline ADNP mRNA level in CTCs shows potential as a prognostic marker for NMIBC with a sensitivity of 83.33% and a specificity of 73.58%. In comparison to baseline, ADNP mRNA expression increased post 2nd TURBT in 5 patients, where 2 experienced recurrence. Meanwhile, among the 12 patients with decreased levels, only one patient relapsed. A considerable limitation of this study entails the small sample size. The Immuno-magnetic beads-ddPCR technique provided a viable method for ADNP mRNA detection in CTCs from bladder cancer patients. The preoperative ADNP mRNA level in CTCs was identified as a prognostic indicator for NMIBC. Longitudinal monitoring of ADNP mRNA in CTCs of bladder cancer patients shows promise in evaluating treatment responses and predicting prognosis.

Immune functions of C-type lectins in medical arthropods.

C-type lectins (CTLs) are a family of proteins that contain 1 or more carbohydrate-recognition domains (CRDs) and bind to a broad repertoire of ligands in the presence of calcium ions. CTLs play important roles in innate immune defenses against microorganisms by acting as pattern-recognition receptors (PRRs) for invading pathogens, such as bacteria, viruses, and parasites. After binding to pathogen-associated ligands, CTLs mediate immune responses, such as agglutination, phagocytosis, and the activation of phenol oxidase progenitors, thereby clearing pathogens. CTLs are an evolutionarily conserved family found in almost all vertebrates and invertebrates. Medical arthropods can acquire and transmit a range pathogens through various approaches, such as bloodsucking, lancing, and parasitism, thus infecting humans and animals with related diseases, some of which can be life-threatening. Recent studies have shown that lectins are important components of the arthropod immune system and are essential for the immune responses of arthropods to arthropod-borne pathogens. This article reviews the current understanding of the structure, function, and signaling pathways involved in CTLs derived from important medical arthropods.

Targeting Haemaphysalis longicornis serpin to prevent tick feeding and pathogen transmission.

Serine proteinase inhibitors (serpins), identified from the hard tick Haemaphysalis longicornis of China, play significant roles in various animal physiological processes. In this study, we showed that H. longicornis serpins (Hlserpin-a and Hlserpin-b) were induced during blood-feeding in nymph ticks and exhibited anticoagulation activity in vitro. Silencing Hlserpins through RNA interference (RNAi) significantly impaired tick feeding. Immunization of mice with recombinant Hlserpins or passive transfer of Hlserpin antiserum significantly curtails the efficacy of tick feeding. Concurrently, the transmission of the Langat virus (LGTV) from ticks to mice witnessed a substantial decrease when Hlserpins were silenced. Our findings suggest that inhibiting Hlserpins can hamper tick engorgement and pathogen transmission, indicating the potential of Hlserpins as a vaccine to counter tick-borne diseases.

Matrix metalloproteinase 3 restricts viral infection by enhancing host antiviral immunity.

Viral pandemics pose great threats to human health and the economy. The host evolved a complex immune response against viral infection. Matrix metalloproteinase 3 (MMP3), also known as stromelysin-1, has an emerging role in immune regulation during pathogen infection. Using in vitro and in vivo infection models, we showed that MMP3 exhibits broad-spectrum antiviral activities against vesicular stomatitis virus (VSV), influenza A virus (H1N1) and human herpes virus 1 (HSV-1). MMP3 deficient mice are susceptible to viral infection and display a compromised antiviral immune response. Correspondingly, the mice with MMP3 overexpression are resistant to viral infection. The mechanistic study suggested that MMP3 is translocated from the cytoplasm into the cell nucleus upon virus infection and influence NF-κB activities, thus amplifying antiviral immune responses. This study suggested a novel function of MMP3 in viral infection and provided new ideas for developing antiviral drugs based on modulating MMP activity.

Multifunctional Clip Strand for the Regulation of DNA Strand Displacement and Construction of Complex DNA Nanodevices.

Strand displacement reactions are important bricks for the construction of various DNA nanodevices, among which the toehold-mediated strand displacement reaction is the most prevalently adopted. However, only a limited number of tools could be used to finely regulate the toehold reaction, thus restricting DNA nanodevices from being more multifunctional and powerful. Herein, we developed a regulation tool, Clip, and achieved multiple regulatory functions, including subtle adjustment of the reaction rates, allosteric strand displacement, selective activation, and resetting of the reaction. Taking advantages of the multiple functions, we constructed Clip-toehold-based DNA walking machines. They showed behaviors of controllable walking, concatenation, and programmable pathways. Furthermore, we built Clip-toehold-based AND and OR logic gates and integrated those logic gates to construct multilayer circuits, which could be reset and reused to process different input signals. We believe that the proposed Clip tool has expanded the functionality of DNA strand displacement-based nanodevices to a much more complex and diverse level and anticipate that the tool will be widely adopted in DNA nanotechnology.

An interlocked DNA cascade system for universal probe-based melting curve analysis.

Single-base mutations are the most common type of mutation in human diseases. Melting curve analysis is currently one of the most commonly used methods to detect single base mutations. However, the existing melting curve analysis cannot possess universality and robust detection performance simultaneously. Therefore, herein, we invented an interlocked DNA cascade system based universal melting curve analysis (ICU-MCA). The strategy is based on the probe dissolution curve method by designing a bridge strand to achieve an ideal distinction between mutant-type DNA and wild-type DNA. What is more, this method can complete multiplexed detection only by changing the bridge sequence, replacing the specific and expensive probe in a traditional probe based melting curve analysis. We performed 6-plex detection on 6 single-base point mutations in BRAC1/2 genes on synthetic single stranded DNA and verified the compatibility of ICU-MCA and PCR and detected BRCA1/c.2082C>T and BRCA2/c.7397T>C mutations in peripheral blood DNA of ovarian cancer patients. Overall, ICU-MCA is one of the best methods in the field of melting curve analysis for detecting single-base mutations.

Eliminating the secondary structure of targeting strands for enhancement of DNA probe based low-abundance point mutation detection.

Nucleic acid probes are very useful tools in biological and medical science. However, the essential sensing mechanism of nucleic acid probes was prone to the interference of surrounding sequences. Especially when the target sequences formed secondary structures such as hairpin or quadruplex, the nucleic acid probes were hindered from hybridizing with target strands, greatly disabled the function of probes. Herein, we have established an Open strand based strategy for eliminating the influence of secondary structures on the performance of nucleic acid probes. The strategy was general toward different lengths, secondary structures and sequences of the targeting strand, and we found that the improvement was higher when the secondary structure of the targeting strand was more complicated. Experiments on synthetic single stranded DNA and real clinical genomic DNA samples were conducted for low abundance mutation detection, and the limit of detection for TERT-C228T and BRCA2 rs80359065 mutations could be 0.02% and 0.05% respectively, demonstrating the clinical practicability of our proposed strategy in low abundance mutation detection.