Target Oligonucleotide Research Articles

Ultrasensitive detection of cancer-associated nucleic acids and mutations by primer exchange reaction-based signal amplification and flow cytometry

The detection of cancer-associated nucleic acids and mutations through liquid biopsy has emerged as a highly promising non-invasive approach for early cancer detection and monitoring. In this study, we report the development of primer exchange reaction (PER) based signal amplification strategy that enables the rapid, sensitive and specific detection of nucleic acids bearing cancer specific single nucleotide mutations using flow cytometry. Using micrometer size beads as support for immobilizing oligonucleotides and programmable PER assembly for target oligonucleotide recognition and fluorescence signal amplification, we demonstrated the versatile detection of target nucleic acids including KRAS oligonucleotide, fragmented mRNAs, and miR-21. Moreover, our detection system can discriminate single base mutations frequently occurred in cancer-associated genes including KRAS, PIK3CA and P53 from cell extracts and circulating tumor DNAs (ctDNAs). The detection is highly sensitive, with a limit of detection down to 27 fM without pre-amplification. In view of a clinical application, we demonstrate the detection of single mutations after extraction and pre-amplification of ctDNAs from the plasma of breast cancer patients. Importantly, our detection strategy enabled the detection of single KRAS mutation even in the presence of 1000-fold excess of wild type (WT) DNA using multi-color flow cytometry detection approach. Overall, our strategy holds immense potential for clinical applications, offering significant improvements for early cancer detection and monitoring.

Complete Genomic Characterization of Global Pathogens, Respiratory Syncytial Virus (RSV), and Human Norovirus (HuNoV) Using Probe-based Capture Enrichment.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in children worldwide, while human noroviruses (HuNoV) are a leading cause of epidemic and sporadic acute gastroenteritis. Generating full-length genome sequences for these viruses is crucial for understanding viral diversity and tracking emerging variants. However, obtaining high-quality sequencing data is often challenging due to viral strain variability, quality, and low titers. Here, we present a set of comprehensive oligonucleotide probe sets designed from 1,570 RSV and 1,376 HuNoV isolate sequences in GenBank. Using these probe sets and a capture enrichment sequencing workflow, 85 RSV positive nasal swab samples and 55 (49 stool and six human intestinal enteroids) HuNoV positive samples encompassing major subtypes and genotypes were characterized. The Ct values of these samples ranged from 17.0-29.9 for RSV, and from 20.2-34.8 for HuNoV, with some HuNoV having below the detection limit. The mean percentage of post-processing reads mapped to viral genomes was 85.1% for RSV and 40.8% for HuNoV post-capture, compared to 0.08% and 1.15% in pre-capture libraries, respectively. Full-length genomes were>99% complete in all RSV positive samples and >96% complete in 47/55 HuNoV positive samples-a significant improvement over genome recovery from pre-capture libraries. RSV transcriptome (subgenomic mRNAs) sequences were also characterized from this data. Probe-based capture enrichment offers a comprehensive approach for RSV and HuNoV genome sequencing and monitoring emerging variants.

Unraveling DNA Triplex Assembly: Mass Spectrometric Investigation of Modified Triplex Forming Oligonucleotides for Enhanced Gene Targeting.

Deoxyribonucleic acid triplexes have potential roles in a range of biological processes involving gene and transcriptional regulation. A major challenge in exploiting the formation of these higher-order structures to target genes in vivo is their low stability, which is dependent on many factors including the length and composition of bases in the sequence. Here, different DNA base modifications have been explored, primarily using native mass spectrometry, in efforts to enable stronger binding between the triplex forming oligonucleotide (TFO) and duplex target sites. These modifications can also be used to overcome pyrimidine interruptions in the duplex sequence in promoter regions of genomes, to expand triplex target sequences for antigene therapies. Using model sequences with a single pyrimidine interruption, triplex forming oligonucleotides containing locked nucleic acid base modifications were shown to have a higher triplex binding propensity than DNA-only and dSpacer-containing TFOs. However, the triplex forming ability of these systems was limited by the competitive formation of multiple higher order assemblies. Triplex forming sequences that correspond to specific gene targets from the Pseudomonas aeruginosa genome were also investigated, with LNA-containing TFOs the only variant able to form triplex using these sequences. This work indicates the advantages of utilizing synthetically modified TFOs to form triplex assemblies in vivo for potential therapeutic applications and highlights the advantages of native mass spectrometry for the study of their formation.

Development of a Novel System Consisting of a Reductase-Like Nanozyme and the Reaction of Resazurin and Ammonia Borane for Sensitive Fluorometric Sensing.

We report a novel system consisting of a redox reaction and a highly efficient reductase-like nanozyme, silica-palladium nanoparticles (Pd@SiO2 NPs), as a novel detection platform for fluorometric sensing. In a proof-of-concept demonstration using an oligonucleotide as the detection target, a glass fiber-based sensor is fabricated by covalently conjugating two oligo probes, which are complementary to the adjacent segments of the target oligonucleotide, on Pd@SiO2 NPs and glass fiber, respectively. In the presence of the target oligonucleotide, the two probes are drawn together by the target through sequence-specific hybridization, bringing the Pd@SiO2 NPs to the glass fiber. When the glass fiber is subsequently immersed in a mixture of resazurin and ammonia borane solution, the Pd@SiO2 NPs on the glass fiber trigger the catalytic conversion of resazurin (blue, slightly fluorescent) to resorufin (pink, highly fluorescent) with massive signal amplification, indirectly signaling the presence of the target oligonucleotide. We show that the glass fiber-based fluorometric sensor can detect a target oligonucleotide associated with the BRAF mutation linearly in the concentration range of 20 to 400 pM with a detection limit (LOD) of 15 pM and the specificity to differentiate targets with single-base difference. These results demonstrate a new frontier for the development of a sensitive, specific, and inexpensive nonenzyme-based fluorometric sensing platform as an alternative to conventional enzyme-based assays.

Separation and Characterization of Therapeutic Oligonucleotide Isomer Impurities by Cyclic Ion Mobility Mass Spectrometry.

Therapeutic oligonucleotides such as antisense oligonucleotide (ASO) and small interfering RNA (siRNA) are among the most remarkable modalities in modern medicine. ASOs and siRNA are composed of single- or double-stranded 15-25 mer synthesized oligonucleotides, which can be used to modulate gene expression. Liquid chromatography-mass spectrometry (LC/MS) is a necessary technique for the quality control of therapeutic oligonucleotides; it is used to evaluate the quantities of target oligonucleotides and their impurities. The widely applied oligonucleotide therapeutic quantitation method uses both ultraviolet (UV) absorbance and the MS signal intensity. Peaks separated from the main peak, which contains full-length product, are generally quantitated by UV. However, coeluting impurities, such as n - 1 shortmers, abasic oligonucleotides, and PS → PO (phosphorothiate to phosphodiester) oligonucleotides, are quantitated by MS. These coeluting impurities can also be comprised of various isomers with the same modification, thus increasing the difficulty in their separation and relative quantitation by LC/MS. It is possible that a specific isomer with a certain structural form induces toxicities. Therefore, characterization of each isomer separation is in high demand. In this study, we separated and characterized oligonucleotide isomers by employing a cyclic ion mobility mass spectrometry (cyclic IMS) system, which allows the separation of ions with the same m/z ratio based on their structural differences. Patisiran antisense and sense strands and their n - 1 and abasic isomers were used as sample sequences, and their ratio characterization was achieved by cyclic IMS. In addition, we evaluated the PS → PO conversion isomers of the antisense strand of givosiran, which originally contained four PS modification sites. The PS → PO isomers exhibited specific and distinguishable mobiligram patterns. We believe that cyclic IMS is a promising method for evaluating therapeutic oligonucleotide isomers.

Lowering an ER stress-regulated long noncoding RNA protects mice from diabetes and isolated pancreatic islets from cell death

We investigated the role of the endoplasmic reticulum (ER) stress-regulated long noncoding-RNA (lncRNA) lncMGC in pancreatic islets and the pathology of type 1 diabetes (T1D), as well as the potential of lncMGC-based therapeutics. In vivo, blood glucose levels (BGL) and HbA1c were significantly lower in lncMGC-knockout (KO)-streptozotocin (STZ)-treated diabetic mice compared to wild-type-STZ. Antisense oligonucleotides (GapmeR) targeting lncMGC significantly attenuated insulitis and BGL in T1D NOD mice compared to GapmeR-negative control (NC). GapmeR-injected T1D Akita mice showed significantly lower BGL compared to Akita-NC mice. hlncMGC-GapmeR lowered BGL in partially humanized lncMGC (hlncMGC)-STZ mice compared to NC-injected mice. CHOP (ER stress regulating transcription factor) and lncMGC were upregulated in islets from diabetic mice but not in lncMGC-KO and GapmeR-injected diabetic mice, suggesting ER stress involvement. In vitro, hlncMGC-GapmeR increased viability of isolated islets from human donors and hlncMGC mice and protected them from cytokine induced apoptosis. Anti-ER stress and anti-apoptotic genes were upregulated, but pro-apoptotic genes were down-regulated in lncMGC KO mice islets and GapmeR-treated human islets. Taken together, these results show that a GapmeR targeting lncMGC is effective in ameliorating diabetes in mice and also preserves human and mouse islet viability, implicating clinical translation potential.

In-vitro Efficacy and In-vivo Biodistribution Studies of an Antisense Oligonucleotide Targeting Leukoencephalopathy with Brainstem and Spinal Cord Involvement and Lactate Elevation (S37.010)

In-vitro Efficacy and In-vivo Biodistribution Studies of an Antisense Oligonucleotide Targeting Leukoencephalopathy with Brainstem and Spinal Cord Involvement and Lactate Elevation (S37.010)

Chondrocyte-Targeted Delivery System of Sortase A-Engineered Extracellular Vesicles Silencing MMP13 for Osteoarthritis Therapy.

Targeted drug delivery and the reduction of off-target effects are crucial for the promising clinical application of nucleic acid drugs. To address this challenge, a new approach for treating osteoarthritis (OA) that accurately delivers antisense oligonucleotides (ASO) targeting matrix metalloproteinase-13 (ASO-MMP13) to chondrocytes, is developed. Small extracellular vesicles (exos) are ligated with chondrocyte affinity peptide (CAP) using Sortase A and subsequently incubated with cholesterol-modified ASO-MMP13 to construct a chondrocyte-targeted drug delivery exo (CAP-exoASO). Compared with exos without CAP (ExoASO), CAP-exoASOs attenuate IL-1β-induced chondrocyte damage and prolong the retention time of ASO-MMP13 in the joint without distribution in major organs following intra-articular injection. Notably, CAP-exoASOs decrease MMP13 expression (P < 0.001) and upregulate COL2A1 expression (P = 0.006), resulting in reorganization of the cartilage matrix and alleviation of progression in the OA model. Furthermore, the Osteoarthritis Research Society International (OARSI) score of articular cartilage tissues treated with CAP-exoASO is comparable with that of healthy rats (P = 0.148). A mechanistic study demonstrates that CAP-exoASO may reduce inflammation by suppressing the IL-17 and TNF signaling pathways. Based on the targeted delivery effect, CAP-exoASOs successfully accomplish cartilage repair and have considerable potential for development as a promising therapeutic modality for satisfactory OA therapy.

Olezarsen for Hypertriglyceridemia in Patients at High Cardiovascular Risk.

Reducing the levels of triglycerides and triglyceride-rich lipoproteins remains an unmet clinical need. Olezarsen is an antisense oligonucleotide targeting messenger RNA for apolipoprotein C-III (APOC3), a genetically validated target for triglyceride lowering. In this phase 2b, randomized, controlled trial, we assigned adults either with moderate hypertriglyceridemia (triglyceride level, 150 to 499 mg per deciliter) and elevated cardiovascular risk or with severe hypertriglyceridemia (triglyceride level, ≥500 mg per deciliter) in a 1:1 ratio to either a 50-mg or 80-mg cohort. Patients were then assigned in a 3:1 ratio to receive monthly subcutaneous olezarsen or matching placebo within each cohort. The primary outcome was the percent change in the triglyceride level from baseline to 6 months, reported as the difference between each olezarsen group and placebo. Key secondary outcomes were changes in levels of APOC3, apolipoprotein B, non-high-density lipoprotein (HDL) cholesterol, and low-density lipoprotein (LDL) cholesterol. A total of 154 patients underwent randomization at 24 sites in North America. The median age of the patients was 62 years, and the median triglyceride level was 241.5 mg per deciliter. The 50-mg and 80-mg doses of olezarsen reduced triglyceride levels by 49.3 percentage points and 53.1 percentage points, respectively, as compared with placebo (P<0.001 for both comparisons). As compared with placebo, each dose of olezarsen also significantly reduced the levels of APOC3, apolipoprotein B, and non-HDL cholesterol, with no significant change in the LDL cholesterol level. The risks of adverse events and serious adverse events were similar in the three groups. Clinically meaningful hepatic, renal, or platelet abnormalities were uncommon, with similar risks in the three groups. In patients with predominantly moderate hypertriglyceridemia at elevated cardiovascular risk, olezarsen significantly reduced levels of triglycerides, apolipoprotein B, and non-HDL cholesterol, with no major safety concerns identified. (Funded by Ionis Pharmaceuticals; Bridge-TIMI 73a ClinicalTrials.gov number, NCT05355402.).

Retroelements in targeted antitumor therapy (review)

Background: Pathological expression of retroelements is determined in most malignant tumors, which reflects the pathogenesis of tumors, since activated retrotransposons cause genomic instability due to a cascade of mutual regulatory influence events between transposons, tumor suppressors and oncogenes. These relationships are reflected in the expression profiles of certain long non-coding RNAs and miRNAs in specific tumor tissues, since retroelements are key sources of non-coding RNAs. Since retroelements serve as drivers for the initiation and progression of carcinogenesis, it is promising to use drugs aimed at inhibiting their expression. The aim of the study: To determine the available methods of influencing retroelements in antitumor therapy, to evaluate the ways of their further improvement for the effective treatment of malignant neoplasms. Materials and methods: The databases Scopus, WoS, PubMed were used to analyze the role of retroelements in the development of tumors, methods of influencing them in the treatment of neoplasms. Results: Reverse transcriptase inhibitors have proven to be effective. Silencing of retrotransposons by acting on histone modifiers can lead to a weakening of the antitumor immune response; therefore, this approach requires RNA guides that direct the formation of heterochromatin in the area where strictly specific retroelements are located. A promising method is viral mimicry aimed at activating the interferon and T-cell response to artificially activated endogenous retroviruses. For this purpose, inhibitors of histone methyltransferases, DNA methyltransferases, and histone deacetylases are used. Their joint use is most effective, especially with PD-1 checkpoint blockers. The arrangement of retroelements in gene introns has become the basis for the development of spliceosomal targeted therapy, which also initiates an antitumor immune response to double-stranded RNA transcripts. We suggest the possibility of using antisense oligonucleotides targeting retroelements in anticancer therapy. Such approach is used to treat age-related macular degeneration, Fukuyama muscular dystrophy, amyotrophic lateral sclerosis. Antisense oligonucleotides targeting microRNAs, proto-oncogenes, and oncosuppressors are used in anticancer therapy and are closely related to retroelements, which could become promising targets for antisense oligonucleotides. Conclusion: In antitumor therapy, it is possible to use two diametrically opposed strategies for influencing retroelements: inhibition and activation. The most promising way is a combination of these methods with specific silencing of retrotransposons using guides (complementary RNAs) and stimulation of the expression of elements not involved in carcinogenesis, but activating the immune response.

Critical Issues on the Surface Functionalization of Plasmonic Au-Ag/TiO2 Thin Films with Thiolated Oligonucleotide-Based Biorecognition Elements.

This work reports on the surface functionalization of a nanomaterial supporting localized surface plasmon resonances (LSPRs) with (synthetic) thiolated oligonucleotide-based biorecognition elements, envisaging the development of selective LSPR-based DNA biosensors. The LSPR thin-film transducers are composed of noble metal nanoparticles (NPs) embedded in a TiO2 dielectric matrix, produced cost-effectively and sustainably by magnetron sputtering. The study focused on the immobilization kinetics of thiolated oligonucleotide probes as biorecognition elements, followed by the evaluation of hybridization events with the target probe. The interaction between the thiolated oligonucleotide probe and the transducer's surface was assessed by monitoring the LSPR signal with successive additions of probe solution through a microfluidic device. The device was specifically designed and fabricated for this work and adapted to a high-resolution LSPR spectroscopy system with portable characteristics. Benefiting from the synergetic characteristics of Ag and Au in the form of bimetallic nanoparticles, the Au-Ag/TiO2 thin film proved to be more sensitive to thiolated oligonucleotide binding events. Despite the successful surface functionalization with the biorecognition element, the detection of complementary oligonucleotides revealed electrostatic repulsion and steric hindrance, which hindered hybridization with the target oligonucleotide. This study points to an effect that is still poorly described in the literature and affects the design of LSPR biosensors based on nanoplasmonic thin films.

A novel mouse model of familial combined hyperlipidemia and atherosclerosis.

Within the context of residual cardiovascular risk in post-statin era, emerging evidence from epidemiologic and human genetic studies have demonstratedthattriglyceride (TG)-rich lipoproteinsand their remnants are causally related to cardiovascular risk. While,carriers of loss-of-function mutations of ApoC3 havelow TG levelsand are protected from cardiovascular disease (CVD). Of translational significance,siRNAs/antisense oligonucleotide (ASO) targeting ApoC3 is beneficial for patients with atheroscleroticCVD. Therefore, animal models of atherosclerosiswith bothhypercholesterolemia and hypertriglyceridemia are important for the discovery of novel therapeutic strategies targeting TG-lowering on top of traditional cholesterol-lowering.In this study, we constructed a novel mouse model of familial combined hyperlipidemia through inserting a human ApoC3 transgene(hApoC3-Tg) into C57BL/6 J mice and injecting a gain-of-function variant of adeno-associated virus-proprotein convertase subtilisin/kexin type 9 (AAV-PCSK9)-D377Y concurrently with high cholesterol diet (HCD) feeding for 16 weeks. In the last 10 weeks, hApoC3-Tg mice were orally treated with a combination of atorvastatin (10 mg·kg-1·d-1) and fenofibrate (100 mg·kg-1·d-1). HCD-treated hApoC3-Tg mice demonstrated elevated levels of serum TG, total cholesterol (TC) and low density lipoprotein-cholesterol (LDL-C). Oral administration of atorvastatin and fenofibrate significantly decreased the plaque sizes of en face aorta, aortic sinus and innominate artery accompanied by improved lipid profileand distribution. In summary,this novel mouse model is of considerable clinical relevance for evaluation of anti-atherosclerotic drugsby targeting both hypercholesterolemia and hypertriglyceridemia.

CircHIPK3 regulates fatty acid metabolism through miR-637/FASN axis to promote esophageal squamous cell carcinoma

The oncogenic role of circRNA in cancers including esophageal cancer (EC) has been well studied. However, whether and how circRNAs are involved in cancer cell metabolic processes remains largely unknown. Here, we reported that circRNA, circHIPK3, is highly expressed in ESCC cell lines and tissues. Knockdown of circHIPK3 significantly restrained cell proliferation, colony formation, migration, and invasion in vitro and inhibited tumor growth in vivo. Mechanistically, circHIPK3 was found to act as a ceRNA by sponging miR-637 to regulate FASN expression and fatty acid metabolism in ESCC cells. Anti-sense oligonucleotide (ASO) targeting circHIPK3 substantially inhibited ESCC both in vitro and in vivo. Therefore, these results uncover a modulatory axis constituting of circHIPK3/miR-637/FASN may be a potential biomarker and therapeutic target for ESCC in the clinic.

Use of Therapeutic RNAs to Accelerate Wound Healing in Diabetic Rabbit Wounds.

Introduction: Diabetes mellitus (DM) affects over 422 million people globally. Patients with DM are subject to a myriad of complications, of which diabetic foot ulcers (DFUs) are the most common with ∼25% chance of developing these wounds throughout their lifetime. Innovation: Currently there are no therapeutic RNAs approved for use in DFUs. Use of dressings containing novel layer-by-layer (LbL)-formulated therapeutic RNAs that inhibit PHD2 and miR-210 can significantly improve diabetic wound healing. These dressings provide sustained release of therapeutic RNAs to the wounds locally without systemic side effects. Clinical Problem Addressed: Diabetic foot wounds are difficult to heal and often result in significant patient morbidity and mortality. Materials and Methods: We used the diabetic neuroischemic rabbit model of impaired wound healing. Diabetes was induced in the rabbits with alloxan, and neuroischemia was induced by ligating the central neurovascular bundle of each ear. Four 6-mm full-thickness wounds were created on each ear. A LbL technique was used to conformally coat the wound dressings with chemically modified RNAs, including an antisense oligonucleotide (antimiR) targeting microRNA-210 (miR-210), an short synthetic hairpin RNA (sshRNA) targeting PHD2, or both. Results: Wound healing was improved by the antimiR-210 but not the PHD2-sshRNA. Specific knockdown of miR-210 in tissue as measured by RT-qPCR was ∼8 Ct greater than nonspecific controls, and this apparent level of knockdown (>99%) suggests that delivery to the tissue is highly efficient at the administered dose. Discussion: Healing of ischemic/neuropathic wounds in diabetic rabbits was accelerated upon inhibition of miR-210 by LbL delivery to the wound bed. miR-210 inhibition was achieved using a chemically modified antisense RNA.

Targeting NPM1 Epigenetically Promotes Postinfarction Cardiac Repair by Reprogramming Reparative Macrophage Metabolism.

Reparative macrophages play a crucial role in limiting excessive fibrosis and promoting cardiac repair after myocardial infarction (MI), highlighting the significance of enhancing their reparative phenotype for wound healing. Metabolic adaptation orchestrates the phenotypic transition of macrophages; however, the precise mechanisms governing metabolic reprogramming of cardiac reparative macrophages remain poorly understood. In this study, we investigated the role of NPM1 (nucleophosmin 1) in the metabolic and phenotypic shift of cardiac macrophages in the context of MI and explored the therapeutic effect of targeting NPM1 for ischemic tissue repair. Peripheral blood mononuclear cells were obtained from healthy individuals and patients with MI to explore NPM1 expression and its correlation with prognostic indicators. Through RNA sequencing, metabolite profiling, histology, and phenotype analyses, we investigated the role of NPM1 in postinfarct cardiac repair using macrophage-specific NPM1 knockout mice. Epigenetic experiments were conducted to study the mechanisms underlying metabolic reprogramming and phenotype transition of NPM1-deficient cardiac macrophages. The therapeutic efficacy of antisense oligonucleotide and inhibitor targeting NPM1 was then assessed in wild-type mice with MI. NPM1 expression was upregulated in the peripheral blood mononuclear cells from patients with MI that closely correlated with adverse prognostic indicators of MI. Macrophage-specific NPM1 deletion reduced infarct size, promoted angiogenesis, and suppressed tissue fibrosis, in turn improving cardiac function and protecting against adverse cardiac remodeling after MI. Furthermore, NPM1 deficiency boosted the reparative function of cardiac macrophages by shifting macrophage metabolism from the inflammatory glycolytic system to oxygen-driven mitochondrial energy production. The oligomeric NPM1 recruited histone demethylase KDM5b to the promoter of Tsc1 (TSC complex subunit 1), the mTOR (mechanistic target of rapamycin kinase) complex inhibitor, reduced histone H3K4me3 modification, and inhibited TSC1 expression, which then facilitated mTOR-related inflammatory glycolysis and antagonized the reparative function of cardiac macrophages. The in vivo administration of antisense oligonucleotide targeting NPM1 or oligomerization inhibitor NSC348884 substantially ameliorated tissue injury and enhanced cardiac recovery in mice after MI. Our findings uncover the key role of epigenetic factor NPM1 in impeding postinfarction cardiac repair by remodeling metabolism pattern and impairing the reparative function of cardiac macrophages. NPM1 may serve as a promising prognostic biomarker and a valuable therapeutic target for heart failure after MI.

An update on the therapeutic role of RNAi in NAFLD/NASH.

Unhealthy lifestyles have given rise to a growing epidemic of metabolic liver diseases, including nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). NAFLD often occurs as a consequence of obesity, and currently, there is no FDA-approved drug for its treatment. However, therapeutic oligonucleotides, such as RNA interference (RNAi), represent a promising class of pharmacotherapy that can target previously untreatable conditions. The potential significance of RNAi in maintaining physiological homeostasis, understanding pathogenesis, and improving metabolic liver diseases, including NAFLD, is discussed in this article. We explore why NAFLD/NASH is an ideal target for therapeutic oligonucleotides and provide insights into the delivery platforms of RNAi and its therapeutic role in addressing NAFLD/NASH.

The single strand template shortening strategy improves the sensitivity and specificity of solid-state nanopore detection.

Through controlling the ssDNA product length of rolling circle amplification with AcyNTP, here we develop a nanopore signal enhancement strategy (STSS), which can successfully transfer the short oligonucleotide targets into long ssDNAs with appropriate lengths that can generate significant translocation currents. By labelling the RCA product with tags such as tetrahedral structures and isothermal amplicons, the resolution, signal specificity, and target range of the STSS can be further extended.

Preparation and evaluation of in situ photocleavable mass tags with facile mass variation for matrix-free laser desorption ionization mass spectrometry.

Mass tags have been used for the precise identification, quantification, and characterization of macrobiomolecules and small organic molecules. Existing research has not yet demonstrated the preparation of a series of trityl-based photocleavable mass tags (PMTs) with similar structures but different molecular weights and mass variability. Herein, we introduce the design and synthesis of trityl-based in situ PMTs that generate heterolytic photocleavable cationic species upon laser irradiation. Mass variation of the PMTs was achieved via a simple conjugation reaction in the final step of synthesis. We prepared a series of PMTs with similar structures but different molecular weights and performed organic matrix-free laser desorption/ionization mass spectrometry (LDI MS) analysis. The practical applicability of the PMTs was evaluated by conjugating PMTs to oligonucleotides and utilizing them for detecting specific oligonucleotide targets combined with a mass signal amplification strategy. Quantitative aspects were also evaluated to verify the capability of the mass tags for multiplexed detection and the quantification of targets. The LDI MS analysis clearly demonstrated in situ heterolytic photocleavage that formed trityl cation peaks with high S/N ratios and high sensitivity. We strongly believe that the developed mass tags and LDI MS are useful alternatives to conventional signal transduction methods used for biosensors, such as surface plasmon resonance, electrochemical redox, and fluorescence.

An anti-PD-1 antisense oligonucleotide promotes the expression of soluble PD-1 by blocking the interaction between SRSF3 and an exonic splicing enhancer of PD-1 exon 3

PD-1 is a key immune checkpoint molecule. Anti-PD-1 immunotherapy is encouraging in cancer treatment. However, it still needs to be improved. PD-1 has at least five isoforms generated by alternative splicing. An isoform without exon 3 encoding soluble PD-1 (sPD-1) can activate anti-tumor immunity by inhibiting the interaction between cellular surface full-length PD-1 (flPD-1) and PD-L1. However, the regulatory mechanism of exon 3 splicing remains largely unknown. Here, we screened the exon 3 sequence by mutation and searched corresponding splicing factors by SpliceAid database and pulldown assay. The alternative splicing of PD-1 exon 3 was analyzed by RT-PCR. The expression levels of flPD-1 and sPD-1 were analyzed by Western blot, flow cytometry, and ELISA. We discovered that an exonic splicing enhancer (ESE) of exon 3 is essential for its inclusion. Moreover, SRSF3 can bind to this ESE and enhance exon 3 inclusion and flPD-1 expression. We designed and screened out an antisense oligonucleotide (ASO) targeting PD-1 to block the interaction between SRSF3 and ESE, and significantly increase exon 3 skipping and sPD-1 expression, which was verified in various tumor cells in addition to oral cancer cells. Altogether, our results uncovered the regulatory mechanism of human PD-1 exon 3 splicing and sPD-1 expression and further designed a novel anti-PD-1 ASO, which are useful for developing a new method of anti-cancer immunotherapy.

Development of simple purification method for oligonucleotides synthesized using phosphoramidite for 5′-end modification as capping reagent

Oligonucleotide therapeutics are generally prepared via solid-phase synthesis, and many impurities derived from the synthesis process are present in crude oligonucleotides. Among the various impurities typical of oligonucleotide synthesis, shortmers, which lack a few residues from the desired oligonucleotide therapeutics, are difficult to remove using conventional high-performance liquid chromatography (HPLC) purification because they have similar structures and physical properties to the desired oligonucleotide. In this study, we developed a simple purification method for oligonucleotide therapeutics. In our strategy, the retention of shortmers on the reversed-phase HPLC column was increased by using a highly lipophilic phosphoramidite, which is a reagent for 5′-end modification of oligonucleotides, instead of acetic anhydride during solid-phase synthesis, and only the target oligonucleotide could be eluted from a reversed-phase HPLC pretreatment column.