Z-DNA is a left-handed double-helical form of DNA that plays roles in transcription, immune responses, viral infection, bacterial biofilm formation, and autoimmune diseases. Despite its importance, the instability of Z-DNA under physiological conditions has hindered detailed structural and functional investigations. Moreover, although antibodies are known to recognize nucleic acids, the mechanisms underlying their detection and stabilization of dynamic DNA under biological conditions remain unclear. This study provides the first atomic-level structural insights into antibody-mediated B-to-Z DNA transition. Accordingly, a Z-DNA-specific chimeric Fab fragment of Z22 (cZ22-Fab) was designed and characterized using multiple biophysical approaches. cZ22-Fab mediates a concentration-dependent B-to-Z conformational transition in CG-repeat DNA, establishing a stable 2:1 Fab/DNA stoichiometry. Crystal structure of cZ22-Fab/Z-DNA complexes revealed a left-handed DNA backbone-tracking recognition mode, in which cZ22-Fab recognizes Z-DNA conformation through phosphate-clamping and base interactions. Notably, a 5′-end C-hanging Z-DNA duplex structure formed by dC(GC)3 and stabilized by cZ22-Fab was observed. Structure-guided mutagenesis demonstrated that heavy chain residues R50 and Y106 are critical for Z-DNA binding, and analyses of additional Z-DNA-forming sequences further elucidated the binding characteristics. Overall, this work provides molecular insights into the mechanism of antibody-mediated Z-DNA formation and stabilization, highlighting its therapeutic relevance and implications for autoimmunity.

Naja atra , the Chinese cobra, is a major cause of snake envenomation in Asia, causing hundreds of thousands of clinical incidents annually. The current treatment, horse serum-derived antivenom, has unpredictable side effects and presents manufacturing challenges. This study focused on developing new-generation snake venom antidotes by using microbial phage display technology to derive nanobodies from an alpaca immunized with attenuated N. atra venom. Following confirmation of the immune response in the alpaca, we amplified V _H H genes from isolated peripheral blood mononuclear cells and constructed a phage display V _H H library of 1.0 × 10 ⁷ transformants. After four rounds of biopanning, the enriched phages exhibited increased binding activity to N. atra venom. Four nanobody clones with high binding affinities were selected: aNAH1, aNAH6, aNAH7, and aNAH9. Specificity testing against venom from various snake species, including two Southeast Asian cobra species, revealed nanobodies specific to the genus Naja . An in vivo mouse venom neutralization assay demonstrated that all nanobodies prolonged mouse survival and aNAH6 protected 66.6% of the mice from the lethal dosage. These findings highlight the potential of phage display-derived nanobodies as valuable antidotes for N. atra venom, laying the groundwork for future applications in snakebite treatment. IMPORTANCE: Chinese cobra venom bites present a formidable medical challenge, and current serum treatments face unresolved issues. Our research applied microbial phage display technology to obtain a new, effective, and cost-efficient treatment approach. Despite interest among scientists in utilizing this technology to screen alpaca antibodies against toxins, the available literature is limited. This study makes a significant contribution by introducing neutralizing antibodies that are specifically tailored to Chinese cobra venom. We provide a comprehensive and unbiased account of the antibody construction process, accompanied by thorough testing of various nanobodies and an assessment of cross-reactivity with diverse snake venoms. These nanobodies represent a promising avenue for targeted antivenom development that bridges microbiology and biotechnology to address critical health needs.

Enterovirus 71 (EV71) is the major etiological agent contributing to the development of hand-foot-mouth disease (HFMD). There are not any global available vaccines or antibody drugs against EV71 released yet. In this study, we perform the virus immunization in a cost-effective and convenient approach by preparing virus particles from size exclusion and immunization of chicken. Polyclonal yolk-immunoglobulin (IgY) was simply purified from egg yolk and monoclonal single-chain variable fragments (scFv) were selected via phage display technology with two scFv libraries containing 6.0 × 10⁶ and 1.3 × 10⁷ transformants. Specific clones were enriched after 5 rounds of bio-panning and four identical genes were classified after the sequence analysis. Moreover, the higher mutation rates were revealed in the CDR regions, especially in the CDR3. IgY showed specific binding activities to both EV71-infected and Coxsackievirus 16-infected cell lysates and high infectivity inhibitory activity of EV71. However, while IgY detected a 37 kDa protein, the selected scFv seemingly detected higher size proteins which could be cell protein instead of EV71 proteins. Despite the highly effective chicken antibody generation, the purity of virus particles prepared by size exclusion is the limitation of this study, and further characterization should be carried out rigorously.