Gang Fang | PubMed Health

PubMed2026

Barcoded oligonucleotide system (BOLT) for targeted organ delivery.

The therapeutic potential of oligonucleotides (oligos) is limited by insufficient delivery to extrahepatic tissues. In vitro assays often fail to accurately predict in vivo behavior, while testing each oligo candidate in animals remains inherently low throughput. Here, we conceive a barcoded oligonucleotide system (BOLT), a platform that enables high-throughput in vivo evaluations of small-molecule ligands and identifies tissue-specific oligo delivery. BOLT integrates rational design of oligo barcodes, modular conjugation chemistry, and next-generation sequencing (NGS)-based quantification, allowing simultaneous evaluation of many chemically diverse ligand-oligo conjugates within a single animal. Notably, this platform is applicable in both mice and nonhuman primates (NHPs). Using BOLT, we discovered ligands with tropism for tissues such as the brain, lung, and muscle. Collectively, these results indicate that the BOLT platform can accelerate the discovery of tissue-targeting ligands for broad oligo therapeutics.

Journal Article↗

PubMed2026

A single DNA methylation site regulates cell fate during Clostridioides difficile sporulation.

DNA methylation is a widespread phenomenon in bacteria that can regulate gene expression, although the mechanisms underlying this epigenetic regulation are often poorly understood. In Clostridioides difficile, the orphan DNA methyltransferase CamA promotes sporulation, a process critical for the persistence and transmission of this nosocomial pathogen. However, the specific CamA target genes that drive this increased sporulation phenotype were unknown. Here, we show that methylation of a single CamA motif in the promoter region of spoIIE, which encodes a factor critical for activating the early-acting sporulation sigma factor, σF, is sufficient to promote spoIIE transcription, σF activation, and spore formation. Surprisingly, the CamA-dependent increase in spoIIE expression also increases the frequency with which cells prematurely activate σF prior to asymmetric division, resulting in miscompartmentalized σF activity. While this premature activation event triggers cell lysis in the well-studied spore-former Bacillus subtilis, we show that C. difficile cells retain developmental plasticity: predivisional cells that have prematurely activated σF can abort sporulation and resume vegetative growth, whereas cells that activate σF in the forespore after asymmetric division remain committed to sporulation. Thus, DNA methylation controls a critical cell fate decision in C. difficile without compromising its capacity to adapt to fluctuating environmental conditions. Finally, we show that CamA confers a significant fitness advantage during murine infection through mechanisms largely independent of its ability to promote sporulation. Since CamA is specific to C. difficile and epigenetically regulates multiple pathways critical for pathogen persistence, these analyses imply that CamA could be a promising antimicrobial target.

DNA Methylation↗