ACS Chemical Biology最新文献

Noncoding RNAs account for up to 98% of the human transcriptome. It has become increasingly clear that noncoding RNAs play diverse and critical roles in many important cellular functions. Although modulation of noncoding RNAs using small molecules is a promising therapeutic strategy, there are relatively few well-characterized RNA-ligand structures. Therefore, the structure-interaction relationships of RNA-targeting small molecules remain underexplored. Here, we present a fragment-based screening approach using biophysical assays to identify and evaluate fragments that bind to the theophylline-binding RNA aptamer, which we use as a model system. We were able to identify high affinity fragment hits and generate models of RNA-ligand complexes using a combination of biophysical data and computational docking. Together, these findings provided insights into the RNA-fragment interactions that underpin binding. This approach demonstrates the feasibility of identifying high-affinity RNA-targeting small molecules with limited structural information.

DNA interstrand cross-links (ICLs) covalently link complementary DNA strands and represent one of the most cytotoxic forms of DNA damage. While their impact on free DNA has been extensively characterized, how ICLs influence nucleosomes─the fundamental units of chromatin─remains largely unexplored. Here, we generated nucleosomes containing site-specific ICLs using click chemistry and systematically examined their effects on nucleosome structure, dynamics, and transcription. Biophysical assays revealed that ICLs did not impair nucleosome assembly, DNA accessibility, or ATP-dependent sliding, and only slightly reduced nucleosome stability. However, in vitro transcription assays demonstrated that ICLs function as absolute barriers to RNA polymerase elongation, producing truncated transcripts that terminate precisely at the cross-linking site in both free DNA and nucleosomal contexts. Restriction enzyme protection assays further showed that transcription-induced nucleosome translocation was unaffected, indicating that arrest results from the inability of the elongation complex to separate cross-linked strands rather than impaired nucleosome mobility. These findings provide mechanistic insight into ICL-induced cytotoxicity at the chromatin level and establish site-specific cross-linked nucleosomes as valuable tools for probing DNA damage responses.

Our ability to respond to emerging pandemics and pathogen resistance relies critically on our ability to build vaccines quickly and efficiently. In this report we used an efficient enzymatic oxidative coupling reaction to create a viral capsid-based vaccine platform that is modular and quickly adaptable for many different pathogens. Tyrosinase-mediated oxidative coupling was used to conjugate C-terminal tyrosine residues on peptide antigens to cysteine residues installed inside MS2 viral capsids. This strategy is particularly promising because the capsids protect the internally conjugated peptides from protease degradation before they are delivered into cells. The vaccine constructs were tested for MHC presentation followed by T-cell activation. Mutants of the MS2 capsid itself activated DC2.4 cells, serving as an adjuvant to help induce the immune response to delivered antigens. The MS2-peptide constructs were shown to be stable in serum, activate DC2.4 cells, and lead to MHC presentation of peptide antigens with subsequent activation of antigen-specific T-cell hybridomas. Taken together, these results demonstrate effective activation of the adaptive immune system in vitro. This synthetic platform can be used to build new vaccines for many different diseases for which immunodominant peptide antigens are known because the antigens can be quickly interchanged while the MS2 scaffold remains the same. Additionally, this platform allows for multiple peptide antigens to be delivered simultaneously in each capsid, which could provide enhanced immunity against resistant pathogen strains and be useful for cancer vaccine development.

Therapies that stimulate DLX5-driven osteogenesis in bone-marrow-derived mesenchymal stem cells (BMSCs) with bone morphogenetic proteins (BMPs) potently accelerate the healing of delayed union and nonunion fractures, but their superior osteoinductive activity is often offset by severe adverse effects. To provide a safer and more effective alternative, we engineered a circular aptamer-antisense oligonucleotide chimera (CircApt-ASO) to activate DLX5-regulated osteogenesis by silencing STAT5A, a key negative regulator of DLX5. CircApt-ASO utilizes a transferrin receptor 1 (TFR1)-binding aptamer, enabling both specific nanoaffinity targeting of BMSCs and efficient intracellular delivery of the anti-STAT5A ASO. Compared with chemically modified linear aptamer-ASO chimeras, CircApt-ASO chimeras exhibit superior biostability and more robust STAT5A gene silencing but negligible cytotoxicity relative to liposome-based ASO delivery methods. Importantly, we demonstrated that CircApt-ASO drastically activated the expression of DLX5 and its downstream osteogenesis-related genes in dose- and time-dependent manners, leading to markedly enhanced osteogenic differentiation. The high stability, potent osteoinductive activity, and minimal cytotoxicity of CircApt-ASO highlight its strong therapeutic potential for promoting bone regeneration in conditions such as delayed union and nonunion fractures.

The Watson–Crick-Franklin (WCF) rules describing nucleobase pairing in antiparallel strands of DNA and RNA can be exploited to create artificially expanded genetic information systems (AEGIS) with as many as 12 independently replicable nucleotides joined by six hydrogen bond pairing schemes. One of these additional pairs joins two nucleotides trivially designated as Z (6-amino-5-nitro-(1H)-pyridin-2-one) and P (2-amino-imidazo-[1,2-a]-1,3,5-triazin-(8H)-4-one). The Z:P pair has supported 6-nucleotide PCR to give diagnostics products, in environmental surveillance kits, and for laboratory in vitro evolution (LIVE) that has generated, inter alia, molecules that inactivate toxins, antibody analogs that bind cancer cells, therapeutic candidates that deliver drugs to those cells, reagents to identify targets on those cells’ surfaces, reagents to move cargoes across the blood–brain barrier, and catalysts with ribonuclease activity. However, the Z nucleoside is acidic, with a pK_a of ∼7.8. In its deprotonated form, Z^– forms a WCF pair with G. This leads to the slow replacement of Z:P pairs by C:G pairs during PCR or, in the reverse process, their introduction. Here, we examine analogs of Z that retain the same donor:donor:acceptor hydrogen bonding pattern as earlier generations of the Z heterocycle, still form a WCF pair with P, but have a higher pK_a. Experiments with Taq polymerase show that the rate of loss of Z:P pairs decreases markedly as the pK_a of the Z heterocycle increases. This provides direct support for the hypothesis that Z:P pairs are in fact lost via deprotonated Z^–:G mismatches. Further, it provides a Z:P system that can be replicated with very high fidelity, with >97% retention of the Z:P pairs over 10,000-fold amplification.

A set of modified 5-methyl- and 5-ethylpyrimidine (uracil and cytosine) and 7-methyl-, 7-ethyl-, and 7-unsubstituted 7-deazapurine (deazaadenine and deazaguanine) ribonucleoside triphosphates was synthesized and used for enzymatic synthesis of base-modified RNA using in vitro transcription (IVT). They all were good substrates for T7 RNA polymerase in the IVT synthesis of model 70-mer RNA, mRNA encoding Renilla luciferase, and 99-mer single-guide RNA (sgRNA). The effect of modifications in the particular RNA on the stability and efficiency in in vitro and in cellulo translation as well as in CRISPR-Cas9 gene cleavage was quantified. In the in vitro translation assay, we observed moderately enhanced luciferase production with 5-methyluracil and -cytosine, while any 7-deazaadenines completely inhibited the translation. Surprisingly, in cellulo experiments showed a significant enhancement of translation with mRNA containing 7-deazaguanine and moderate enhancement with 5-methyl- or 5-ethylcytosine. Most of the modifications had a minimal effect on the efficiency of the gene cleavage in CRISPR-Cas9 except for 7-alkyl-7-deazaadenines that completely inhibited the cleavage. The results are important for further design of potential base-modified RNA therapeutics.

Hexokinase domain containing protein 1 (HKDC1) is a recently discovered fifth human hexokinase isozyme that is significantly upregulated in several disease states, including lung and liver cancers. Cellular studies suggest that HKDC1 is a low activity hexokinase; however, its functional characteristics have remained enigmatic. Here, we describe the kinetic and regulatory features of recombinant human HKDC1, demonstrating it to be a robust hexokinase (k_cat/K_m,glucose = 1.5 × 10⁴ M^–1 s^–1) with a unique glucose K_m value (0.49 ± 0.07 mM) that differs markedly from all other human hexokinase isozymes. The isolated C-terminal domain of HKDC1 displays kinetic characteristics nearly identical to the full-length enzyme, whereas the N-terminal domain is inactive. Unlike all other 100 kDa vertebrate hexokinases characterized to date, HKDC1 is insensitive to product inhibition by physiological concentrations of glucose 6-phosphate, with apparent inhibition constants above 1 mM. The hexokinase activity of HKDC1 is also insensitive to Dinaciclib, a pan cyclin-dependent kinase inhibitor that reportedly disrupts the ability of nuclear localized HKDC1 to phosphorylate retinoblastoma-binding protein 5. Conversely, the hexokinase activity of HKDC1 is potently inhibited by a synthetic glucosamine derivative previously developed for hexokinase 1 and 2, with an IC₅₀ value of 103 ± 6 nM. An HKDC1 variant associated with retinitis pigmentosa, T58M, displays a modest, but statistically significant 2-fold decrease in catalytic efficiency (k_cat/K_m,glucose) compared to the wild-type enzyme. Together, our results provide a detailed functional characterization of recombinant HKDC1 and set the stage for investigating the link between HKDC1 catalysis and human disease.

Immunomodulatory imide drugs (IMiDs), including thalidomide, lenalidomide, and pomalidomide, can be used to induce degradation of a protein of interest that is fused to a short degron motif, which often comprises a zinc finger (ZF). These IMiDs, however, also induce the degradation of endogenous ZF-containing neosubstrates, including IKZF1, IKZF3, and SALL4. To improve degradation selectivity, we took a bump-and-hole approach to design and screen bumped IMiD analogues against 8380 ZF mutants. This yielded a bumped IMiD analogue that induces efficient degradation of a mutant ZF degron, while not affecting other cellular proteins, including IKZF1, IKZF3, and SALL4. In proof-of-concept studies, this system was applied to induce degradation of the optimum degron fused to CDK9, HPRT1, NanoLuc, or TRIM28. We anticipate that this system will be a valuable addition to the current arsenal of degron systems for use in target validation.

Capnine-like sulfonolipids are sulfonate-containing analogs of sphingolipids found in many Bacteroidetes bacteria, where they govern essential functions such as gliding motility, outer membrane polysaccharide assembly, and antibiotic susceptibility. In gut-associated anaerobic Bacteroidetes, these sulfonolipids also modulate host–microbe interactions. In aerobic bacteria, the capnine precursor cysteate is produced by a pyridoxal phosphate (PLP)-dependent cysteate synthase (CapA1), a close homologue of cystathionine β-synthase (CBS). By contrast, the mechanism of cysteate production in anaerobic Bacteroidetes bacteria has not been biochemically studied. Herein, we report the characterizations of archaeal cysteate synthase homologue from the anaerobic bacteria Alistipes finegoldii (AfCapA2). Biochemical assays confirm its ability to catalyze the conversion of O-phosphoserine (OPS) to cysteate. Crystal structures of AfCapA2 in complex with PLP and OPS-PLP identify essential catalytic residues and reveal a structural similarity to threonine synthase, unlike CapA1, which is more similar to CBS. Comparative analysis of CapA1 and this nonorthologous CapA2, including structural differences, catalytic versatility, and phylogenetic distribution across Bacteroidetes, suggests convergent evolution of cysteate synthase activity. Our work clarifies the details of sulfonolipid synthesis in anaerobic bacteria and the biochemical origins of this structurally distinctive lipid in the gut microbiome.