Chemically modified nucleic acids have become a powerful platform for basic research and applied technologies. Universal nucleobases are used in PCR, sequencing, and the design of nanodevices and aptamers. Fluorescent universal nucleobases have an even wider range of applications, including the development of nucleic acid-based sensors, switches, and relay logic gates. However, few such nucleobases have been proposed to date, with most having suboptimal optical properties such as low brightness and fluorescence quenching by flanking nucleobases. Here, we propose an adenine-based molecular rotor, 7,8-dihydro-8-oxo-6-(3-methylbenzo[d]thiazol-2(3H)-ylidene)adenine (oxo-AdeBZT), as a new, remarkably bright and potent fluorescent nucleobase. Its brightness in both oligodeoxyribonucleotides (ODNs) and DNA duplexes (4200-10 000 M-1 cm-1) originates from a high molar extinction coefficient (averaged ε368 37 000 M-1 cm-1), provided by the appended 3-methylbenzo[d]thiazolyl moiety, and a relatively high quantum yield (0.11-0.27). Melting temperature and Gibbs free energy variations observed upon the incorporation of oxo-AdeBZT opposite various native nucleobases in a duplex context typically did not exceed 5 °C and 0.6 kcal mol-1, respectively. The basis of these uniform hybridization properties was unveiled using computational methods. According to molecular dynamics simulations, oxo-AdeBZT pushes the opposite nucleobase out of the DNA double helix and forms multiple hydrophobic contacts with the flanking base pairs. At the same time, the rotational mobility of the bonds between the oxo-AdeBZT-constituting heterobicycles decreases, and oxo-AdeBZT adopts a planar conformation in both ODNs and their duplexes, resulting in the light-up effect. These properties make oxo-AdeBZT a promising molecular tool for analytical, biophysical and biochemical studies.

Enhanced matrix assisted laser desorption ionization mass spectrometry (eMALDI MS) refers to MALDI MS in which the efficacy of desorption and ionization is enhanced by heat, volatile species, or both, generated from chemical decomposition of additives in the sample of analyte under MALDI conditions. MALDI MS has been widely used for oligonucleotide (ON) analysis. However, two challenges, limited ability to analyze long ONs and low tolerance to trace salts, significantly restrict its applications. We recently proposed the ion pair thermal model for the gas phase ion formation mechanism of MALDI MS. This model predicts an exponential increase in ion yield with plume temperature. Building on this model, we investigated the effects of azobisisobutyronitrile (AIBN), which decomposes under MALDI conditions and generates heat and volatile species, on MALDI performance. We found that the resulting eMALDI MS method provides more reliable analysis of long ONs and exhibits greater tolerance to alkaline salts.

The guanine O6 phosphitylation side reaction, which can lead to the formation of the intermediates - abasic sites and O6 phosphate derivatives - responsible for internucleotide bond cleavage and G-to-2,6-diaminopurine (D), or G-to-A after PCR amplification, substitution error, respectively, represents a significant barrier to the synthesis of long oligonucleotides (ONs) and limits the use of phosphoramidite capping in the synthesis of sensitive ONs. To confirm this side reaction and determine the degree to which it occurs, we synthesized the sequence 5'-T6GT18-3' and subjected its 5'-capped version to repeated coupling and oxidation reaction cycles. Subsequent deprotection and cleavage with neat nBuNH2 and LCMS analysis of the resulting products confirmed that both intermediates can form simultaneously, with quantities increasing with the increase of the number of reaction cycles. In addition, after a sufficient number of cycles, a substantial amount of thymine O4 phosphate derivative, which can cause T-to-C substitution errors and has not been reported in the literature, was also observed.

Fluorescent nucleobase analogs (FBAs) are widely used probes for nucleic acid analysis. In this study, we designed, synthesized, and investigated fluorescent 7-arylethenyl-7-deazaguanosine derivatives as novel FBAs conjugated to molecular rotor structures. More than 10 types of oligonucleotides (ONs) containing the designed FBAs were constructed via the postsynthetic modification of 7-iodo-7-deazaguanosine using Suzuki-Miyaura cross-coupling. This approach allowed the efficient construction of a guanosine-type FBA library. The duplex stability of ONs containing 7-arylethenyl-7-deazaguanosine was slightly lower than that of canonical ONs. In contrast, naphthylethenyl-modified derivatives unexpectedly showed enhanced triplex stability compared with canonical DNA triplexes. The synthesized FBAs exhibited fluorescence emissions in the visible region, and the fluorescence spectra of the duplexes and triplexes containing 7-arylethenyl-7-deazaguanosine exhibited enhanced emissions, which varied depending on the aryl substituent. In particular, upon G·TA triplex formation, 1-or 2-naphthylethenyl-modified derivatives acted as a molecular rotor and showed strong fluorescence enhancement in a sequence-selective manner.

Development of multiple heartcutting two-dimensional liquid chromatography with ion-pairing reversed-phase and hydrophilic interaction separations for analysis of impurities in therapeutic oligonucleotides.

ApoE-directed CpG nano-immunoadjuvant ameliorates Alzheimer's-like pathology in mice.

Carrier-free spherical nucleic acids engineered by coordination-competition for programmable release and cancer immunotherapy.

Determination of adversity and the no observed adverse effect level (NOAEL) in nonclinical toxicity studies are important components in the overall assessment of the potential for human risk during pharmaceutical development. Resources exist to guide adversity decisions in general, for findings in ganglia of the peripheral nervous system (PNS), and for gliosis in the central nervous system (CNS). The objective of this opinion article is to describe considerations that may apply when determining the adversity (or lack thereof) of nerve fiber degeneration (NFD) in either the CNS or PNS following intrathecal (IT) administration of oligonucleotide (ON) therapeutics (such as antisense oligonucleotides and short interfering RNAs). It is important to differentiate IT delivery procedure-related NFD effects from ON-related effects or ON-related exacerbation of procedure-related findings. This article presents evidence-based approaches for deciding whether NFD associated with IT ON administration is an adverse microscopic finding in the brain, spinal cord, spinal nerve roots, ganglia, and/or nerves. In the authors' collective experience, nonextensive (i.e., regionally restricted), minimal or mild NFD unaccompanied by functional or behavioral deficits, neuronal necrosis, or a substantial inflammatory response in neural tissue can be interpreted as nonadverse.

Modern LC-MS strategies for comprehensive metabolite identification of therapeutic oligonucleotides.

In solid-phase oligonucleotide (ON) synthesis, a capping step is beneficial for obtaining a pure full-length ON. Acetic anhydride (Ac2O) is widely used as a conventional capping reagent. However, several problems associated with acetyl capping have been reported. In this study, we developed a novel phosphoramidite-type capping reagent, ethano N,N-dicyclohexylphosphoramidite (EDCP), which was obtained as an easy-to-handle crystalline solid. EDCP, with a sterically accessible phosphorus atom, exhibited a higher capping ability than Ac2O and diethyl N,N-diisopropylphosphoramidite, which was previously reported as a phosphoramidite-type capping reagent. Furthermore, capping-derived byproducts were not detected during solid-phase ON synthesis using EDCP. These results indicate that EDCP is a useful capping reagent that can serve as an alternative to Ac2O and has potential applications in ON synthesis.

BackgroundThe use of programmable nucleases has transformed genome editing and functional genomics. Clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) was developed such that targeted genomic lesions [usually DNA double-stranded breaks (DSBs)] could be introduced in vivo with ease and precision. In the presence of homology donors, these lesions facilitate high-efficiency precise genome editing (PGE) via homology-directed repair (HDR) pathways. Because DSBs can lead to genomic instability, however, a large amount of effort has been invested in methodologies (e.g., base editors) that only require nicking the chromosomal DNA on one strand. Indeed, we have demonstrated in human cells that oligodeoxynucleotide (ODN)-mediated PGE using nickase variants of Cas9 can proceed by at least two HDR subpathways termed synthesis-dependent strand annealing (SDSA) and single-stranded DNA incorporation (ssDI). Which pathway is utilized is determined by which chromosomal strand (sense or anti-sense/Watson or Crick) is nicked and by the strandedness (sense or anti-sense/Watson or Crick) of the donor ODN.ResultsWhile the mechanism of mammalian SDSA is moderately well understood, that of ssDI is not. To gain genetic insight into ssDI, we carried out a genome-wide CRISPR knockout screen to identify those genes which, when absent, enable increased ssDI. This screen identified the protein lysine methyl transferase (PKMT) Su(var)3–9, enhancer-of-zeste and trithorax (SET) domain bifurcated histone lysine methyltransferase 1 (SETDB1):activating transcription factor 7-interacting protein (ATF7IP) heterodimer and the downstream human silencing hub (HUSH) complex as strong negative regulators of ssDI. Consistent with their well-known biological effects, the negative regulation of ssDI by SETDB1/ATF7IP and HUSH was specific for transgenic reporters and for a HUSH-regulated single-copy gene, but was not observed at other (non-HUSH regulated) single-copy endogenous loci.ConclusionsIn toto, these experiments underscore the profound impact that chromatin modifiers - and by extension, chromatin structure – have on PGE outcomes. Specifically, we have identified SETDB1/ATF7IP and the HUSH complex as major negative regulators of the HDR subpathway, ssDI, when the target is a transgene. These experiments are a proof-of-principle that chromatin can act as a potent barrier to genetic recombination and they strongly support the feasibility of extending similar chromatin modulating strategies to enhance PGE efficiency at endogenous single-copy loci.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13072-026-00661-6.

Pneumonias remain a leading cause of death worldwide. Seeking novel strategies to protect susceptible patients, we have reported that inhaled delivery of a diacylated lipopeptide and a synthetic CpG oligodeoxynucleotide (ODN) protects animals against a broad range of infectious pneumonias by stimulating antimicrobial responses from the lung epithelium. Toll-like receptor 9 (TLR9) is well-established as the primary cellular receptor for CpG ODNs. However, we recently reported that ODNs also stimulate TLR9-independent generation of antimicrobial mitochondrial reactive oxygen species. By testing a variety of synthetic ODN molecules, we found that ODNs containing a phosphorothioate backbone, but not those with a phosphodiester backbone, induce TLR9-independent pathogen killing in lungs and improve mouse survival. Phosphorothioate-backboned ODN binds mitochondrial protein voltage-dependent anion channel 1 (VDAC1) at its N terminus, initiating pneumonia-protective metabolic reprogramming in lung epithelial cells that yield the protective antimicrobial effect. Thus, the phosphorothioate backbone of ODN is a critical structural pattern that activates TLR9-independent, metabolically-modulated innate immune protection that may be harnessed to protect vulnerable patients against pneumonia.

Oligonucleotide (ON) therapeutics are promising as disease-modifying therapies for central nervous system (CNS) disorders. Intrathecal ON administration into the cerebral spinal fluid is a safe and effective delivery mode to the CNS. However, preclinical studies have shown acute and transient changes in neurobehavior following high-dose central ON delivery. Here, we characterize a subset of these changes peaking 15 min after ON dosing and resolving after 120 min. Symptoms include shaking, muscle twitching, cramping, hyperactivity, hyperreactivity, vocalizations, tremors, convulsions, and seizures. These are collectively referred to as the acute neuronal activation response. Acute neuronal activation is observed in rats, mice, and nonhuman primates and is quantifiable using a simple scoring system. It is distinct from acute inhibition seen with some phosphorothioate-modified antisense oligonucleotides, characterized by loss of spinal reflexes, ataxia, and sedation. The acute neuronal activation response is largely sequence-independent and is driven by ON chelation of divalent cations, particularly influenced by the divalent cation-to-ON ratio in the dosing solution. Acute neuronal activation can be safely mitigated by adjusting this ratio through magnesium supplementation in the ON formulation. We provide a comprehensive framework for quantifying and mitigating the acute neuronal activation response caused by high concentrations of centrally delivered ON therapeutics in preclinical species.

Hydrophilic interaction chromatography (HILIC) is an attractive separation mode for the analysis of therapeutic oligonucleotides (ONs). ONs are very polar compounds that are commonly dissolved in highly aqueous media, whereas HILIC eluents often comprise a high percentage of organic solvent. This solvent mismatch can cause breakthrough and peak splitting. In this study, we investigated the effects of the sample solvent composition and injection volume on the HILIC separation of nucleobases and ONs, and to what extent an in-line mixer between injector and column can mitigate breakthrough and peak splitting. Using isocratic HILIC with nucleobases as medium-polar, less-retentive test compounds, we illustrated that an injection solvent of 90% water results in peak broadening, which deteriorates with increasing injection volume, leading to serious peak deformations and asymmetries. Here, in-line mixing did not improve peak shapes, as the mixer volume adds to band broadening and the homogenization of the injection solvent with the HILIC eluent does not lead to on-column focusing conditions. When analyzing an ON mixture (15/16/17-mer) dissolved in a weak solvent (30% water) by gradient HILIC, injection volumes could be increased up to 20µL (5.7% of the column volume) without losing separation performance. However, when the ON mixture was dissolved in 100% water, injection volumes above 2µL caused severe peak distortion and extensive analyte breakthrough in gradient HILIC. When injecting the ONs in 100% water with a 100-µL in-line mixer, no analyte breakthrough and peak splitting were observed for injection volumes up to 40µL, while maintaining baseline separation of the ONs. The usefulness of in-line mixing in HILIC was demonstrated by the analysis of a pharmaceutical antisense ON (ASO). Large-volume injection of this ON in 100% water allowed for the separation and detection of the main compound and adjacent minor impurities, without breakthrough or severe peak distortion.

Decoding diastereomeric and impurity profiles of phosphorothioated siRNAs via 2D anion-exchange chromatography coupled with ion-pair reversed phase liquid chromatography mass spectrometry.

Characterizing the tumor immune microenvironment (TIME) to explore potential therapeutic targets is fundamental to advancing precision tumor immunotherapy. However, the immunosuppressive nature of "cold" tumors, notably prostate cancer, poses a significant barrier to immunotherapy, demanding new approaches to simultaneously reinvigorate anti-tumor immunity and modulate the molecular drivers of immune evasion. Here, we identified VSIG4 as a key regulator of prostate tumor-resident macrophage fate through single-cell sequencing analysis. Meanwhile, a shikonin (Shik)-mediated downregulation of VSIG4 in macrophages is verified, potentially attenuating its immunosuppressive effects. Building on these findings, cytosine guanine dinucleotide (CpG) oligodeoxynucleotide (ODN)-modified manganese (Mn)-Shik metal-polyphenol network nanodrugs (Mn/Shik@CpG NDs) are designed to reverse the "cold" immune environment of prostate tumor. In this scenario, Mn/Shik@CpG NDs release monomeric components under the stimulation of acidic and glutathione-rich tumor microenvironment (TME), thus exerting their immunomodulatory effects synergistically. Since the released Shik can induce DNA damage by necroptosis promoting reactive oxygen species production, cGAS-STING signaling pathway is initiated, which further activates interferon production in the TME. In addition, the necroptosis of Shik initiates immunogenic cell death, further activating innate immunity and promoting adaptive immune responses. Mn2+ is a cGAS-STING sensitizer, which amplifies the intratumoral interferon response. As an immune adjuvant, CpG ODN effectively promotes the maturation of dendritic cells, as well as the helper T cell differentiation and pro-inflammatory cytokine secretion, thus activating both innate and adaptive immunity. In vivo studies suggest that Shik-mediated VSIG4 downregulation, combined with innate and adaptive immune activation, remodels the TIME to evoke a significant anti-tumor response. Furthermore, transcriptomic analysis of rechallenged tumors indicated this durable protection was driven by a genuine immune memory response, revealing a gene signature of T cell activation and immune reprogramming. Collectively, beyond presenting a novel therapeutic candidate for converting immunologically "cold" tumors into "hot" ones, our work validates a data-guided design pipeline, offering a conceptual blueprint to inform the precise engineering of future nanodrugs.

The imbalanced expression of interferon regulatory factor (IRF) 4 and IRF8 in activated B cells significantly influences their differentiation and promotes the development of immune-related diseases. Restoring abnormal B cells to appropriate responses may treat these diseases. In this study, an oligodeoxynucleotide (ODN) S2, designed according to the consensus sequence recognized by IRFs in interferon-stimulated response elements, was used as an immunomodulator to investigate its effects on mouse splenic B cells stimulated with the TLR9 agonist CpG ODN, either alone or in combination with antigen, and to explore its underlying mechanisms. The results showed that S2 had a significant negative regulatory effect on CpG ODN induced B cell activation. It also significantly downregulated the production of IL-6 and the percentage of IL-6+ B cells in splenocytes stimulated by CpG ODN, but significantly upregulated the percentage of IL-10+ B cells. Interestingly, S2 impaired antibody production both in vitro and in vivo, but rescued mice from lethal inflammatory responses. Further studies showed that S2 could bind IRF4 and IRF8 with high affinity, slightly upregulate phosphorylated IRF4, reduce the expression and nuclear translocation of IRF8, and alter the proportion of IRF4+, IRF8+ or double-positive B cells in spleen cells induced by CpG ODN. These results suggest that S2 acts as a decoy directing some B cells to differentiate into IL-10-producing Breg-like cells rather than plasma cells by restoring the TLR9 signal-induced IRF4 and IRF8 ratio imbalance. This indicates its potential as an immunomodulator for the treatment of diseases associated with B-cell abnormalities.

Carbomer-lecithin-based adjuvant Adjuplex has been shownto inducea balanced T cell response and high antibody titers with some of theviral pathogens. Here, we compared the humoral immune response inmice immunized intranasally with respiratory syncytial virus fusion(RSV-F) or glycoprotein (RSV-G) or both using Adjuplex and/or a TLR9agonist, CpG oligodeoxynucleotide (ODN). Both the adjuvants elicitedrobust antibody response in mice; however, Adjuplex generated higherlevels of neutralizing antibodies as compared to CpG ODN when usedwith RSV-F protein. Neutralizing antibody titers were associated withreduced RSV load in the lungs of immunized BALB/c mice upon intranasalchallenge with the RSV A2 strain. RSV-G alone was insufficient toelicit a strong antibody response with either of the adjuvants, andcombining RSV-F and RSV-G did not show any synergistic response interms of neutralizing antibody titers; however, this combination ofantigens elicited a strong mucosal immune response as measured byIgA titers specific to RSV-F and RSV-G in lung tissues. These datasuggest that carbomer-based adjuvants can induce a potent and protectivehumoral and mucosal response when used as an adjuvant for the RSV-Fsubunit vaccine, and the combination of RSV-F and RSV-G with Adjuplexmay further be explored for enhancing the efficacy of RSV vaccines.

Oligonucleotides (ONs) have gained significant attention as therapeutic agents, due to their ability to selectively target genetic sequences. As ON-based drug development expands, analytical methods are essential to ensure quality and purity. While ion pairing reversed phase liquid chromatography (IP-RPLC) is the gold standard for ON impurities characterization, alternative chromatographic modes are increasingly used. However, fully resolving all impurities with a single one-dimensional method remains challenging. Comprehensive two-dimensional liquid chromatography offers improved impurity profiling by combining orthogonal separations. However, differences in elution strength between the fraction collected from the first dimension (1D) and the mobile phase used in the second dimension (2D) can cause peak distortion and breakthrough phenomena. Interestingly, under certain conditions, these distortion effects can disappear, yielding a breakthrough peak accompanied by a symmetrical retained peak. This phenomenon, referred to as "total breakthrough," was recently described in 2D-LC separations of small molecules and peptides. Although the underlying mechanisms are not yet fully elucidated, the total breakthrough (TB) strategy provides an advantage in 2D-LC, by enabling the use of large injection volumes, effectively overcoming injection-related issues commonly caused by solvent incompatibility between dimensions. In this study, we first demonstrated that ONs exhibit TB behavior in IP-RPLC. Upon confirming this behavior, we selected a 2D HILIC × IP-RPLC configuration (a combination often considered challenging due to limited solvent compatibility) and performed an in-depth investigation of both 1D HILIC collected fraction and 2D IP-RPLC conditions affecting breakthrough behavior. We found that understanding TB of ONs in IP-RPLC is more complex than with classical small molecules, primarily due to the interplay between ion-pairing agents in the 2D -mobile phase and residual salts originating from 1D Through careful optimization, we successfully established a comprehensive HILIC × IP-RPLC method that enabled analysis of ONs mixture, taking advantage of the TB behavior of ONs, while preserving sufficient sensitivity.

Because of their pivotal role in liver fibrosis, activated hepatic stellate cells (aHSCs) may serve as a promising target for innovative medical treatments. Endoplasmic reticulum stress activation through inositol-requiring enzyme1 (IRE1)-X-box-binding protein-1 (XBP1) is a significant event associated with hepatic stellate cells (HSC) activation. We evaluated the potential impact of treatment with XBP1-specific decoy oligodeoxynucleotide (ODN) on modulation of aHSC. To activate HSCs, LX-2 cells were treated with transforming growth factor β (5 ng/mL). Meanwhile, the sequence of XBP1-specific decoy ODN was designed using the JASPAR (open-access transcription factor binding profile data base) and CLC Main Workbench (Qiagen) software. The outcome of treatment with ODN on aHSC was analyzed using quantitative reverse transcription polymerase chain reaction, immunoblotting, scratch assay, and ELISA. Transfection of activated LX-2 cells with 1 μg XBP1 decoy ODN downregulated the expression level of lysyl oxidase, tissue inhibitor of matrix metalloproteinase, α-smooth muscle actin, and fibronectin genes. In addition, the immunoblotting analysis and ELISA assay showed that XBP1 decoy ODN significantly reduced protein expression of α-smooth muscle actin and collagen secretion, respectively, compared to control cells. Our research may lead to innovative treatments for liver fibrosis, providing hope for better outcomes for patients with this chronic condition. SIGNIFICANCE STATEMENT: This study applied a novel decoy oligodeoxynucleotide targeting X-box-binding protein-1 to suppress the activation of hepatic stellate cells, a key driver of liver fibrosis. By modulating endoplasmic reticulum stress and fibrogenic gene expression, this strategy offers a promising therapeutic avenue for chronic liver diseases.