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We developed a system to detect multiple target biomolecules through sensing motif-tethered oligodeoxynucleotides. DNA-based molecular probes gave the primary amine motif upon reaction with the target biomolecules, glutathione (GSH) and H 2 O 2 . After labelling with biotin, the product DNAs were selectively collected to be quantified by qPCR.

The interaction between glioma cells and astrocytes promotes the proliferation of gliomas. Micro-RNAs (miRNAs) carried by astrocyte exosomes (exos) may be involved in this process, but the mechanism remains unclear. The oligonucleotide AS1411, which consists of 26 bases and has a G-quadruplex structure, is an aptamer that targets nucleolin. In this study, we demonstrate exosome-miRNA-27a-mediated cross-activation between astrocytes and glioblastoma and show that AS1411 reduces astrocytes' pro-glioma activity. The enhanced affinity of AS1411 toward nucleolin is attributed to its G-quadruplex structure. After binding to nucleolin, AS1411 inhibits the entry of the NF-κB pathway transcription factor P65 into the nucleus, then downregulates the expression of miRNA-27a in astrocytes surrounding gliomas. Then, AS1411 downregulates astrocyte exosome-miRNA-27a and upregulates the expression of INPP4B, the target gene of miRNA-27a in gliomas, thereby inhibiting the PI3K/AKT pathway and inhibiting glioma proliferation. These results were verified in mouse orthotopic glioma xenografts and human glioma samples. In conclusion, the parallel structure of AS1411 allows it to bind to nucleolin and disrupt the exosome-miRNA-27a-mediated reciprocal activation loop between glioma cells and astrocytes. Our results may help in the development of a novel approach to therapeutic modulation of the glioma microenvironment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Various artificial oligodeoxynucleotides (ODNs) that contribute to gene regulation have been developed and their diversity and multifunctionality have been demonstrated. However, few artificial ODNs are actively transported to the cell nucleus, despite the fact that gene regulation also takes place in both the cell nucleus and the cytoplasm. In this study, to prepare ODNs with the ability to accumulate in the cell nucleus, we introduced Hoechst molecules into ODNs that act as carriers of functional molecules to the cell nucleus (Hoe-ODNs). We synthesized Hoe-ODNs and confirmed that they bound strongly to DNA duplexes. When single-stranded Hoe-ODNs or double-stranded ODNs consisting of Hoe-ODNs and its complementary strand were administered into living cells, both ODNs were efficiently accumulated in the cell nucleus. In addition, antisense ODNs, which were tethered with Hoechst unit, were delivered into the cell nucleus and efficiently suppressed the expression of their target RNA. Thus, we constructed a delivery system that enables the transport of ODNs into cell nucleus., (© 2023 Wiley-VCH GmbH.)

Framework nucleic acids (FNAs) of various morphologies, designed using the precise and programmable Watson-Crick base pairing, serve as carriers for biomolecule delivery in biology and biomedicine. However, the impact of their shape, size, concentration, and the spatial presentation of cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODNs) on immune activation remains incompletely understood. In this study, representative FNAs with varying morphologies are synthesized to explore their immunological responses. Low concentrations (50 nM) of all FNAs elicited no immunostimulation, while high concentrations of elongated DNA nanostrings and tetrahedrons triggered strong activation due to their larger size and increased cellular uptake, indicating that the innate immune responses of FNAs depend on both dose and morphology. Notably, CpG ODNs' immune response can be programmed by FNAs through regulating the spatial distance, with optimal spacing of 7-8 nm eliciting the highest immunostimulation. These findings demonstrate FNAs' potential as a designable tool to study nucleic acid morphology's impact on biological responses and provide a strategy for future CpG-mediated immune activation carrier design., (© 2023 Wiley-VCH GmbH.)

Lumican is an extracellular matrix proteoglycan known to regulate toll-like receptor (TLR) signaling in innate immune cells. In experimental settings, lumican suppresses TLR9 signaling by binding to and sequestering its synthetic ligand, CpG-DNA, in non-signal permissive endosomes. However, the molecular details of lumican interactions with CpG-DNA are obscure. Here, the 3-D structure of the 22 base-long CpG-DNA (CpG ODN_2395) bound to lumican or TLR9 were modeled using homology modeling and docking methods. Some of the TLR9-CpG ODN_2395 features predicted by our model are consistent with the previously reported TLR9-CpG DNA crystal structure, substantiating our current analysis. Our modeling indicated a smaller buried surface area for lumican-CpG ODN_2395 (1803 Å 2 ) compared to that of TLR9-CpG ODN_2395 (2094 Å 2 ), implying a potentially lower binding strength for lumican and CpG-DNA than TLR9 and CpG-DNA. The docking analysis identified 32 amino acids in lumican LRR1-11 interacting with CpG ODN_2395, primarily through hydrogen bonding, salt-bridges, and hydrophobic interactions. Our study provides molecular insights into lumican and CpG-DNA interactions that may lead to molecular targets for modulating TLR9-mediated inflammation and autoimmunity.

Whole-genome synthesis provides a powerful approach for understanding and expanding organism function 1-3 . To build large genomes rapidly, scalably and in parallel, we need (1) methods for assembling megabases of DNA from shorter precursors and (2) strategies for rapidly and scalably replacing the genomic DNA of organisms with synthetic DNA. Here we develop bacterial artificial chromosome (BAC) stepwise insertion synthesis (BASIS)-a method for megabase-scale assembly of DNA in Escherichia coli episomes. We used BASIS to assemble 1.1 Mb of human DNA containing numerous exons, introns, repetitive sequences, G-quadruplexes, and long and short interspersed nuclear elements (LINEs and SINEs). BASIS provides a powerful platform for building synthetic genomes for diverse organisms. We also developed continuous genome synthesis (CGS)-a method for continuously replacing sequential 100 kb stretches of the E. coli genome with synthetic DNA; CGS minimizes crossovers 1,4 between the synthetic DNA and the genome such that the output for each 100 kb replacement provides, without sequencing, the input for the next 100 kb replacement. Using CGS, we synthesized a 0.5 Mb section of the E. coli genome-a key intermediate in its total synthesis 1 -from five episomes in 10  days. By parallelizing CGS and combining it with rapid oligonucleotide synthesis and episome assembly 5,6 , along with rapid methods for compiling a single genome from strains bearing distinct synthetic genome sections 1,7,8 , we anticipate that it will be possible to synthesize entire E. coli genomes from functional designs in less than 2 months., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Atherosclerosis is a progressive chronic inflammatory condition that is the cause of most cardiovascular and cerebrovascular diseases. The transcription factor nuclear factor‑κB (NF‑κB) regulates a number of genes involved in the inflammatory responses of cells that are critical to atherogenesis, and signal transducer and activator of transcription (STAT)3 is a key transcription factor in immunity and inflammation. Decoy oligodeoxynucleotides (ODNs) bind to sequence‑specific transcription factors and limit gene expression by interfering with transcription in vitro and in vivo . The present study aimed to investigate the beneficial functions of STAT3/NF‑κB decoy ODNs in liposaccharide (LPS)‑induced atherosclerosis in mice. Atherosclerotic injuries of mice were induced via intraperitoneal injection of LPS and the mice were fed an atherogenic diet. Ring‑type STAT3/NF‑κB decoy ODNs were designed and administered via an injection into the tail vein of the mice. To investigate the effect of STAT3/NF‑κB decoy ODNs, electrophoretic mobility shift assay, western blot analysis, histological analysis with hematoxylin and eosin staining, Verhoeff‑Van Gieson and Masson's trichrome staining were performed. The results revealed that STAT3/NF‑κB decoy ODNs were able to suppress the development of atherosclerosis by attenuating morphological changes and inflammation in atherosclerotic mice aortae, and by reducing pro‑inflammatory cytokine secretion through inhibition of the STAT3/NF‑κB pathway. In conclusion, the present study provided novel insights into the antiatherogenic molecular mechanism of STAT3/NF‑κB decoy ODNs, which may serve as an additional therapeutic intervention to combat atherosclerosis.

In the field of nucleic acid nanotechnology and therapeutics, there is an imperative need to improve the oligodeoxynucleotides' (ODNs) properties by either chemical modification of the oligonucleotides' structure or to covalently link them to a reporter or therapeutic moieties that possess biologically relevant properties. The chemical conjugation can thus significantly improve the intrinsic properties not only of ODNs but also reporter/therapeutic molecules. Bioconjugation of nucleic acids to small molecules also serves as a nano-delivery facility to transport various functionalities to specific targets. Herein, we describe a generalized methodology that deploys azide-alkyne cycloaddition, a click reaction to conjugate a cyanine-3 alkyne moiety to an azide-functionalized ODN 12-mer, as well as 3-azido 7-hydroxycoumarin to an alkyne functionalized ODN 12-mer., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Knock-in mice are useful for evaluating endogenous gene expressions and functions in vivo. Instead of the conventional gene-targeting method using embryonic stem cells, an exogenous DNA sequence can be inserted into the target locus in the zygote using genome-editing technology. In this chapter, I describe the generation of epitope-tagged mice using engineered endonuclease and single-strand oligodeoxynucleotide through the mouse zygote as an example of how to generate a knock-in mouse by genome editing., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Acne vulgaris has a pathogenesis that involves increased sebum production and perifollicular inflammation. Sterol regulatory element-binding protein-1 (SREBP-1) and peroxisome proliferator activated receptor-γ (PPAR-γ) are transcription factors that regulate numerous genes involved in lipid biosynthesis. To improve a new therapeutic approach, we designed the SREBP/PPAR decoy oligodeoxynucleotide (ODN), a synthetic short DNA containing complementary sequences for the SREBP and PPAR transcription factors. We aim to investigate the beneficial functions and the molecular mechanisms of the synthetic SREBP/PPAR decoy ODN in lipogenic models. C. acnes was intradermally injected with a 1.0 × 10 7 colony forming unit/20 μL. The synthetic SREBP/PPAR decoy ODN or scrambled decoy ODN (10 μg) was transferred via the mouse tail vein injection. SZ95 cells were transfected with 2 μg of synthetic ODNs. After transfection, the SZ95 cells were cultured in serum-free medium containing 20 ng/μL of insulin-like growth factor-1 (IGF)-1 for 24 h. To investigate the expression of gene and signaling pathways, we performed Western blotting. The distribution of the chimeric decoy ODN was confirmed by EMSA. Lipid levels were assessed by Nile red and Oil Red O staining. The cytokine levels were measured by ELISA kit. This study showed that C. acnes -injected mice and IGF-1-stimulated SZ95 cells exhibited increased expression of SREBP-1 and PPAR-γ compared to the normal controls. In contrast, the administration of the SREBP/PPAR chimeric decoy ODN significantly suppressed the upregulation of lipogenic genes. Furthermore, the SREBP/PPAR decoy ODN decreased the plasma cytokines and cytokine levels of total protein. These results suggested that the SREBP/PPAR decoy ODN exerts its anti-lipogenic effects by regulating lipid metabolism and by inhibiting lipogenesis through the inactivation of the SREBP and PPAR pathways. Therefore, the synthetic SREBP/PPAR ODN demonstrates substantial therapeutic feasibility for the treatment of acne vulgaris.

CpG Oligodeoxynucleotides (ODNs) are established TLR9 ligands; however, their functional responses in CD4+ T cells are believed to be independent of TLR9 and MyD88. We studied ligand-receptor interactions of ODN 2216 and TLR9 in human CD4+ T cells and assessed their consequences in terms of TLR9 signalling and cell phenotype. We demonstrated that the uptake of ODN 2216, a synthetic TLR9 agonist, is controlled by TLR9 signalling molecules and results in an increase in the expression of TLR9 signalling molecules, regulated via a feedback mechanism. Next, the uptake of ODN 2216 resulted in TLR9 signalling dependent but MyD88 independent increase in expression of TGF-β. Finally, ODN 2216 treated CD4+ T cells showed an anti-inflammatory phenotype that was similar to Th3 type of regulatory T cells. These Th3-like cells were able to suppress the proliferation of untreated CD4+ T cells. Collectively, our results demonstrate a direct and interdependent relationship between ODN 2216 uptake and TLR9 signalling in CD4+ T cells. Our findings thus pave the way for future research to explore direct modulation of adaptive immune cells, using innate immune ligands, to subvert exaggerated inflammatory responses., Competing Interests: CONFLICT OF INTEREST The authors declare no conflict of interest.

As a method of gene therapy, application of decoy oligodeoxynucleotides (ODNs) could interfere at the pretranscription level, by blocking the transcription factors, and inhibiting their attachment to the corresponding sequences in genomic DNA. Some of the transcription factors including MYC, OCT4, SOX2, STAT3, and NANOG are associated with the stemness properties of cancer cells, and suppressing them could interfere with cellular differentiation, which synergizes the efficiency of other anticancer therapies. The use of decoy ODNs has shown to be an effective measure against various malignancies, and it has shown to have a synergic effect when it is used along with the other cancer therapy methods. Emergence of modern nanocarriers has shown to further improve the outcome of using decoy ODNs against some cancers, and it has the potential of being used for clinical applications. In this chapter, it was aimed to provide a glance of this method for cancer therapy., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Various diseases, including cancer, are caused by genetic mutations. A 5'-tailed duplex (TD) DNA, consisting of a long single-stranded (ss) editor DNA and a short (∼35-base) ss assistant oligodeoxyribonucleotide, can introduce a base-substitution in living cells and thus correct mutated genes. Previously, several hundred-base DNAs were employed as the editor DNAs. In this study, 5'-TDs were prepared from various editor DNAs with different lengths and examined for their gene correction abilities, using plasmid DNA bearing a mutated copepod green fluorescent protein (copGFP) gene, in human cells. High-throughput analysis was performed by the reactivated fluorescence of the wild-type protein encoded by the corrected gene as the indicator. The analysis revealed that 5'-TDs with ∼100-base ss editor DNAs enabled gene editing at least as efficiently as those with longer editor DNAs. Moreover, the antisense strand was more effective as the editor than the sense strand, in contrast to the 5'-TDs with longer editor strands. These results indicated that the 5'-TD fragments with shorter editor strands than those used in previous studies are useful nucleic acids for gene correction., (Copyright © 2021 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Purpose of Review: Molecular forms of allergen-specific immunotherapy (AIT) are continuously emerging to improve the efficacy of the treatment, to shorten the duration of protocols and to prevent any side effects. The present review covers the recent progress in the development of AIT based on nucleic acid encoding allergens or CpG oligodeoxynucleotides (CpG-ODN)., Recent Findings: Therapeutic vaccinations with plasmid deoxyribonucleic acid (DNA) encoding major shrimp Met e 1 or insect For t 2 allergen were effective for the treatment of food or insect bite allergy in respective animal models. DNA expressing hypoallergenic shrimp tropomyosin activated Foxp3+ T regulatory (Treg) cells whereas DNA encoding For t 2 down-regulated the expression of pruritus-inducing IL-31. Co-administrations of major cat allergen Fel d 1 with high doses of CpG-ODN reduced Th2 airway inflammation through tolerance induction mediated by GATA3+ Foxp3hi Treg cells as well as early anti-inflammatory TNF/TNFR2 signaling cascade. Non-canonical CpG-ODN derived from Cryptococcus neoformans as well as methylated CpG sites present in the genomic DNA from Bifidobacterium infantis mediated Th1 or Treg cell differentiation respectively., Summary: Recent studies on plasmid DNA encoding allergens evidenced their therapeutic potential for the treatment of food allergy and atopic dermatitis. Unmethylated or methylated CpG-ODNs were shown to activate dose-dependent Treg/Th1 responses. Large clinical trials need to be conducted to confirm these promising preclinical data. Moreover, tremendous success of messenger ribonucleic acid (mRNA) vaccines against severe acute respiratory syndrome coronavirus 2 must encourage as well the re-exploration of mRNA vaccine platform for innovative AIT., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Aptamers offer a great opportunity to develop innovative drug delivery systems that can deliver cargos specifically into targeted cells. In this study, a chimera consisting of two aptamers was developed to deliver doxorubicin into cancer cells and release the drug in cytoplasm in response to adenosine-5'-triphosphate (ATP) binding. The chimera was composed of the AS1411 anti-nucleolin aptamer for cancer cell targeting and the ATP aptamer for loading and triggering the release of doxorubicin in cells. The chimera was first produced by hybridizing the ATP aptamer with its complementary DNA sequence, which is linked with the AS1411 aptamer via a poly-thymine linker. Doxorubicin was then loaded inside the hybridized DNA region of the chimera. Our results show that the AS1411-ATP aptamer chimera was able to release loaded doxorubicin in cells in response to ATP. In addition, selective uptake of the chimera into cancer cells was demonstrated using flow cytometry. Furthermore, confocal laser scanning microscopy showed the successful delivery of the doxorubicin loaded in chimeras to the nuclei of targeted cells. Moreover, the doxorubicin-loaded chimeras effectively inhibited the growth of cancer cell lines and reduced the cytotoxic effect on the normal cells. Overall, the results of this study show that the AS1411-ATP aptamer chimera could be used as an innovative approach for the selective delivery of doxorubicin to cancer cells, which may improve the therapeutic potency and decrease the off-target cytotoxicity of doxorubicin.

Single-stranded oligodeoxynucleotides (ssODNs) are widely used as DNA repair templates in CRISPR/Cas precision genome editing. However, the underlying mechanisms of single-strand templated DNA repair (SSTR) are inadequately understood, constraining rational improvements to precision editing. Here we study SSTR at CRISPR/Cas12a-induced DNA double-strand breaks (DSBs) in the eukaryotic model green microalga Chlamydomonas reinhardtii. We demonstrate that ssODNs physically incorporate into the genome during SSTR at Cas12a-induced DSBs. This process is genetically independent of the Rad51-dependent homologous recombination and Fanconi anemia pathways, is strongly antagonized by non-homologous end-joining, and is mediated almost entirely by the alternative end-joining enzyme polymerase θ. These findings suggest differences in SSTR between C. reinhardtii and animals. Our work illustrates the promising potentially of C. reinhardtii as a model organism for studying nuclear DNA repair., (© 2021. The Author(s).)

A simple carbon nanodot-based electrogenerated chemiluminescence biosensor is described for sensitive and selective detection of microRNA-21 (miRNA-21), a biomarker of several pathologies including cardiovascular diseases (CVDs). The photoluminescent carbon nanodots (CNDs) were obtained using a new synthesis method, simply by treating tiger nut milk in a microwave reactor. The synthesis is environmentally friendly, simple, and efficient. The optical properties and morphological characteristics of the CNDs were exhaustively investigated, confirming that they have oxygen and nitrogen functional groups on their surfaces and exhibit excitation-dependent fluorescence emission, as well as photostability. They act as co-reactant agents in the anodic electrochemiluminescence (ECL) of [Ru(bpy) 3 ] 2+ , producing different signals for the probe (single-stranded DNA) and the hybridized target (double-stranded DNA). These results paved the way for the development of a sensitive ECL biosensor for the detection of miRNA-21. This was developed by immobilization of a thiolated oligonucleotide, fully complementary to the miRNA-21 sequence, on the disposable gold electrode. The target miRNA-21 was hybridized with the probe on the electrode surface, and the hybridization was detected by the enhancement of the [Ru(bpy) 3 ] 2+ /DNA ECL signal using CNDs. The biosensor shows a linear response to miRNA-21 concentration up to 100.0 pM with a detection limit of 0.721 fM. The method does not require complex labeling steps, and has a rapid response. It was successfully used to detect miRNA-21 directly in serum samples from heart failure patients without previous RNA extraction neither amplification process., (© 2021. The Author(s).)

I-motif or C4 is a four-stranded DNA structure with a protonated cytosine:cytosine base pair (C+:C) found in cytosine-rich sequences. We have found that oligodeoxynucleotides containing adenine and cytosine repeats form a stable secondary structure at a physiological pH with magnesium ion, which is similar to i-motif structure, and have named this structure 'adenine:cytosine-motif (AC-motif)'. AC-motif contains C+:C base pairs intercalated with putative A+:C base pairs between protonated adenine and cytosine. By investigation of the AC-motif present in the CDKL3 promoter (AC-motifCDKL3), one of AC-motifs found in the genome, we confirmed that AC-motifCDKL3 has a key role in regulating CDKL3 gene expression in response to magnesium. This is further supported by confirming that genome-edited mutant cell lines, lacking the AC-motif formation, lost this regulation effect. Our results verify that adenine-cytosine repeats commonly present in the genome can form a stable non-canonical secondary structure with a non-Watson-Crick base pair and have regulatory roles in cells, which expand non-canonical DNA repertoires., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Small interfering RNA (siRNA) is the most important tool for the manipulation of mRNA expression and needs protection from intracellular nucleases when delivered into the cell. In this work, we examined the effects of siRNA modification with the phosphoryl guanidine (PG) group, which, as shown earlier, makes oligodeoxynucleotides resistant to snake venom phosphodiesterase. We obtained a set of siRNAs containing combined modifications PG/2'-O-methyl (2'-OMe) or PG/2'-fluoro (2'-F); biophysical and biochemical properties were characterized for each duplex. We used the UV-melting approach to estimate the thermostability of the duplexes and RNAse A degradation assays to determine their stability. The ability to induce silencing was tested in cultured cells stably expressing green fluorescent protein. The introduction of the PG group as a rule decreased the thermodynamic stability of siRNA. At the same time, the siRNAs carrying PG groups showed increased resistance to RNase A. A gene silencing experiment indicated that the PG-modified siRNA retained its activity if the modifications were introduced into the passenger strand.

In biological systems, conformational changes and allosteric modulation play pivotal roles in regulating biological functions, such as the dynamic change of protein molecules, in response to binding or interacting with other factors such as pH, voltage, salt, light, or ligand. RNA can be manipulated and tuned with a level of simplicity that is characteristic of DNA or polymers, while displaying versatility in structure, diversity in function, and adaptability in a configuration similar to proteins. In the past, the work on the investigation of conformational change mainly focused on protein. The induced-fit and conformational capture in RNA have also been explored, such as in the study of riboswitches. Herein, we report the engineering of three-dimensional RNA nanocubes and demonstrated the operation and regulation for its configuration. We demonstrate the operation of reconfigurable RNA nanocubes whose shapes change precisely and reversibly in response to a specific trigger strand. The shape, size, and conformation can be regulated precisely and reversibly in response to the specific triggering signals. The shape and conformational conversion were observed by cryo-EM and gel electrophoresis, respectively. Harnessing the size, shape, conformation, and self-assembly capabilities of the RNA nanocube can provide a new potential use of this technology as nanocarriers for the treatment of various diseases., (© 2021 Blanco Carcache et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

A C-nucleoside derivative of phenylpyridine or the respective palladacycle was incorporated at either 3'- or 5'-terminus of a short oligodeoxynucleotide. Hybridization properties of these modified oligonucleotides were studied in a fluorescence-based competition assay in addition to conventional UV melting temperature analysis and compared with those of a previously prepared analogue featuring the modified nucleoside in the middle of the sequence. With the unpalladated phenylpyridine oligonucleotides, UV melting temperature qualitatively correlated with the ability to displace a strand from a double helix in the competition assay, decreasing in the order 5' > 3' > middle. Corresponding results on the palladacyclic oligonucleotides were more difficult to interpret but both UV melting and competition experiments revealed a decrease in the duplex stability upon palladation in most cases. On the other hand, dependence of the UV melting temperature on the identity of the canonical nucleobase opposite to the modified nucleobase analogue was much more pronounced with the palladacyclic duplexes than with their unpalladated counterparts. Furthermore, UV melting profiles of the palladacyclic duplexes featured an additional transition at a temperature exceeding the melting temperature of the unmodified part of the duplex. Taken together, these results lend support to the idea of Pd(II)-mediated base pairs that are highly stable but incompatible with the geometry of a double helix., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

We present Triplex-seq, a deep-sequencing method that systematically maps the interaction space between an oligo library of ssDNA triplex-forming oligos (TFOs) and a particular dsDNA triplex target site (TTS). We demonstrate the method using a randomized oligo library comprising 67 million variants, with five TTSs that differ in guanine (G) content, at two different buffer conditions, denoted pH 5 and pH 7. Our results show that G-rich triplexes form at both pH 5 and pH 7, with the pH 5 set being more stable, indicating that there is a subset of TFOs that form triplexes only at pH 5. In addition, using information analysis, we identify triplex-forming motifs (TFMs), which correspond to minimal functional TFO sequences. We demonstrate, in single-variant verification experiments, that TFOs with these TFMs indeed form a triplex with G-rich TTSs, and that a single mutation in the TFM motif can alleviate binding. Our results show that deep-sequencing platforms can substantially expand our understanding of triplex binding rules and aid in refining the DNA triplex code.

Mechanical force applied by ultrasound in solution leads to the dissociation of DNA metallo-base-pair interactions when these motifs are functionalized with oligodeoxynucleotide sequences of sufficient length. The annealing and force-induced denaturing process is followed by the attachment of distance-sensitive fluorescent probes and is found to be reversible.

In multicellular organisms, expression profiling in spatially defined regions is crucial to elucidate cell interactions and functions. Here, we establish a transcriptome profiling method coupled with photo-isolation chemistry (PIC) that allows the determination of expression profiles specifically from photo-irradiated regions of interest. PIC uses photo-caged oligodeoxynucleotides for in situ reverse transcription. PIC transcriptome analysis detects genes specifically expressed in small distinct areas of the mouse embryo. Photo-irradiation of single cells demonstrated that approximately 8,000 genes were detected with 7 × 10 4 unique read counts. Furthermore, PIC transcriptome analysis is applicable to the subcellular and subnuclear microstructures (stress granules and nuclear speckles, respectively), where hundreds of genes can be detected as being specifically localised. The spatial density of the read counts is higher than 100 per square micrometre. Thus, PIC enables high-depth transcriptome profiles to be determined from limited regions up to subcellular and subnuclear resolutions., (© 2021. The Author(s).)

Genome-wide identification of DNA double-strand breaks (DSBs) induced by CRISPR-associated protein (Cas) systems is vital for profiling the off-target events of Cas nucleases. However, current methods for off-target discovery are tedious and costly, restricting their widespread applications. Here we present an easy alternative method for CRISPR off-target detection by tracing the integrated oligonucleotide Tag using next-generation-sequencing (CRISPR-Tag-seq, or Tag-seq). Tag-seq enables rapid and convenient profiling of nuclease-induced DSBs by incorporating the optimized double-stranded oligodeoxynucleotide sequence (termed Tag), adapters, and PCR primers. Moreover, we employ a one-step procedure for library preparation in Tag-seq, which can be applied in the routine workflow of a molecular biology laboratory. We further show that Tag-seq successfully determines the cleavage specificity of SpCas9 variants and Cas12a/Cpf1 in a large-scale manner, and discover the integration sites of exogenous genes introduced by the Sleeping Beauty transposon. Our results demonstrate that Tag-seq is an efficient and scalable approach to genome-wide identification of Cas-nuclease-induced off-targets.

Ly49Q is an ITIM-bearing MHC class I receptor that is highly expressed in plasmacytoid dendritic cells (pDCs). Ly49Q is required for the TLR9-mediated IFN-I production in pDCs, although the mechanism is not fully understood. We here demonstrate that Ly49Q protects pDCs from pyroptotic cell death induced by CpG oligodeoxynucleotides (CpG). In the Ly49Q-deficient (Klra17 -/- ) mouse spleen, the number of ssDNA-positive pDCs increased significantly after CpG treatment, strongly suggesting that Klra17 -/- pDCs were susceptible to CpG-induced cell death. In Klra17 -/- bone-marrow-derived dendritic cells (BMDCs), CpG-induced cell death was accompanied by increased cathepsin B leakage from the vesicular compartments into the cytoplasm. Concurrently, IL-1β secretion increased in the CpG-treated Klra17 -/- BMDCs, strongly suggesting that the CpG-induced cell death in these cells is pyroptotic in nature. Consistent with these observations, inhibiting cathepsin B or caspase 1 in CpG-stimulated Klra17 -/- BMDCs reversed the increase in cell death. Pyroptotic cell death and IL-1β secretion were also observed in BMDCs derived from transgenic mice expressing an ITIM-less Ly49Q (Ly49Q-YF Tg). CpG also increased the IL-1β production and cell death in B2m -/- BMDCs. These results suggest that Ly49Q and MHC class I play important roles for protecting pyroptosis-like cell death of DCs by influencing lysosome state., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Synonymous codon pair deoptimization is an efficient strategy for virus attenuation; however, the underlying mechanism remains controversial. Here, we optimized and deoptimized the codon pair bias (CPB) of the human immunodeficiency virus type 1 (HIV-1) envelope ( env ) gene to investigate the influence of env synonymous CPB recoding on virus replication capacity, as well as the potential mechanism. We found that env CPB deoptimization did not always generate attenuation, whereas CPB optimization attenuated virus replication in MT-4 cells. Furthermore, virus attenuation correlated with reduced Env protein production but not with decreased viral RNA synthesis. Remarkably, in our model, increasing the number of CpG dinucleotides in the 5' end of env did not reduce the replication capacity of HIV-1. These results indicate that factors other than CPB or CpG content may have impacted the viral fitness of the synonymously recoded study variants. Our findings provide evidence that CPB recoding-associated attenuation can affect translation efficiency. Moreover, we demonstrated that an increased number of CpGs in the 5' end of HIV-1 env is not always associated with reduced virus replication capacity.

Signal transducer and activator of transcription 3 (STAT3) is a critical regulator for angiogenesis, cell cycle progression, apoptosis, and drug resistance. Resistance toward EGF receptor (EGFR) inhibitors is a significant clinical concern for metastatic colon cancer patients. The present study aimed to evaluate the blocking influences of STAT3 decoy oligodeoxynucleotides (ODNs) on the STAT3 survival signaling pathway in nonresistant and erlotinib-resistant SW480 colon cancer cells. First, STAT3 decoy and scramble ODNs were designed according to STAT3 elements in the promoter region of MYCT1 gene and tested for the interaction of STAT3 protein with designed ODNs via in silico molecular docking study. Then, the efficiency of transfection and subcellular localization of ODNs were assessed using flow cytometry and fluorescence microscopy, respectively. Cell viability, cell cycle, and apoptosis tests, scratch and colony formation assays, and real-time PCR were also used to study the cancerous properties of cells. A considerable decrease in proliferation of colon cancer cells was observed with blockade of STAT3 signaling due to cell cycle arrest and induced apoptosis via downregulation of cyclin D1 and Bcl-XL, respectively. Furthermore, upon transfecting STAT3 decoy ODNs, colony formation potential and migration activity in both SW480 colon cancer cell lines were decreased compared to the control groups. From this study, it could be concluded that STAT3 is critical for cell growth inhibition and metastatic properties reduction of resistant SW480 colon cancer cells; therefore, STAT3 decoy ODNs could be considered as potential therapeutics along with current remedies for treating drug-resistant colon cancer., (© 2020 International Federation for Cell Biology.)

Type 1 diabetes occurs as a consequence of progressive autoimmune destruction of beta cells. A potential treatment for this disease should address the immune attack on beta cells and their preservation/regeneration. The objective of this study was to elucidate whether the immunomodulatory synthetic oligonucleotide IMT504 was able to ameliorate diabetes in NOD mice and to provide further understanding of its mechanism of action. We found that IMT504 restores glucose homeostasis in a diabetes mouse model similar to human type 1 diabetes, by regulating expression of immune modulatory factors and improving beta cell function. IMT504 treatment markedly improved fasting glycemia, insulinemia, and homeostatic model assessment of beta cell function (HOMA-Beta cell) index. Moreover, this treatment increased islet number and decreased apoptosis, insulitis, and CD45 + pancreas-infiltrating leukocytes. In a long-term treatment, we observed improvement of glucose metabolism up to 9 days after IMT504 cessation and increased survival after 15 days of the last IMT504 injection. We postulate that interleukin (IL)-12B (p40), possibly acting as a homodimer, and Galectin-3 (Gal-3) may function as mediators of this immunomodulatory action. Overall, these results validate the therapeutic activity of IMT504 as a promising drug for type 1 diabetes and suggest possible downstream mediators of its immunomodulatory effect.

The laboratorial diagnosis of the intestinal schistosomiasis is always performed using Kato-Katz technique. However, this technique presents low sensitivity for diagnosis of individuals with low parasite burden, which constitutes the majority in low endemicity Brazilian locations for the disease. The objective of this study was developed and to validate a real-time PCR assay (qPCR) targeting 121 bp sequence to detect Schistosoma spp. DNA for the diagnosis of intestinal schistosomiasis and a sequence of the human β-actin gene as internal control. Firstly, the qPCR was standardized and next it was evaluated for diagnosis and cure assessment of intestinal schistosomiasis in the resident individuals in Tabuas and Estreito de Miralta, two locations in Brazil endemic for intestinal schistosomiasis. The qPCR assay results were compared with those of the Kato-Katz (KK) test, examining 2 or 24 slides, Saline Gradient (SG) and "reference test" (24 KK slides + SG). The cure assessment was measured by these diagnostic techniques at 30, 90, and 180 days post-treatment. In Tabuas, the positivity rates obtained by the qPCR was 30.4% (45/148) and by "reference test" was of 31.0% (46/148), with no statistical difference (p = 0.91). The presumed cure rates at 30, 90, and 180 days post-treatment were 100, 94.4, and 78.4% by the analysis of 24 KK slides, 100, 94.4, and 78.4% by the SG, and 100, 83.3, and 62.1% by the qPCR assay. In Estreito de Miralta, the positivity obtained by qPCR was 18.3% (26/142) and with "reference test" was 24.6% (35/142), with no statistical difference (p = 0.20). The presumed cure rates were 93.3, 96.9, and 96.5% by the KK, 93.3, 96.9, and 100% by the SG, and 93.3, 93.9, and 96.5% by the qPCR at 30, 90, and 180 days post-treatment, respectively. This study showed that the diagnostic techniques presented different performance in the populations from the two districts (Tabuas and Estreito de Miralta) and reinforces the need of combining techniques to improve diagnosis accuracy, increasing the detection of individuals with low parasite burden. This combination of techniques consists an important strategy for controlling the disease transmission., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Siqueira, Senra, de Oliveira, Carneiro, Gomes, Rabello, Coelho and Oliveira.)

Despite recent advances, non-Hodgkin's B cell lymphoma patients often relapse or remain refractory to therapy. Therapeutic resistance is often associated with survival signaling via nuclear factor κB (NF-κB) transcription factor, an attractive but undruggable molecular target. In this study, we describe a bipartite inhibitor comprising a NF-κB-specific decoy DNA tethered to a CpG oligodeoxynucleotide (ODN) targeting Toll-like receptor-9-expressing B cell lymphoma cells. The Bc-NFκBdODN showed efficient uptake by human diffuse large B cell (U2932, OCI-Ly3), Burkitt (RaJi), and mantle cell (Jeko1) lymphomas, respectively. We confirmed that Bc-NFκBdODN inhibited NF-κB nuclear translocation and DNA binding, resulting in CCND2 and MYC downregulation. Bc-NFκBdODN enhanced radiosensitivity of lymphoma cells in vitro. In xenotransplanted human lymphoma, local injections of Bc-NFκBdODN reduced NF-κB activity in whole tumors. When combined with a local 3-Gy dose of radiation, Bc-NFκBdODN effectively arrested OCI-Ly3 lymphoma progression. In immunocompetent mice, intratumoral injections of Bc-NFκBdODN suppressed growth of directly treated and distant A20 lymphomas, as a result of systemic CD8 T cell-dependent immune responses. Finally, systemic administration of Bc-NFκBdODN to mice bearing disseminated A20 lymphoma induced complete regression and extended survival of most of the treated mice. Our results underscore clinical relevance of this strategy as monotherapy and in support of radiation therapy to benefit patients with resistant or relapsed B cell lymphoma., Competing Interests: Declaration of Interests M.K. and P.S. are on the patent application submitted by CoH that covers the design of oligonucleotides presented in this report. The remaining authors declare no competing interests., (Copyright © 2020 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Base pair mismatches in DNA can erroneously be incorporated during replication, recombination, etc. Here, the influence of A…A mismatch in the context of 5'CAA·5'TAG sequence is explored using molecular dynamics (MD) simulation, umbrella sampling MD, circular dichroism (CD), microscale thermophoresis (MST) and NMR techniques. MD simulations reveal that the A…A mismatch experiences several transient events such as base flipping, base extrusion, etc. facilitating B-Z junction formation. A…A mismatch may assume such conformational transitions to circumvent the effect of nonisostericity with the flanking canonical base pairs so as to get accommodated in the DNA. CD and 1D proton NMR experiments further reveal that the extent of B-Z junction increases when the number of A…A mismatch in d(CAA)·d(T(A/T)G) increases (1-5). CD titration studies of d(CAA)·d(TAG) n=5 with the hZα ADAR1 show the passive binding between the two, wherein, the binding of protein commences with B-Z junction recognition. Umbrella sampling simulation indicates that the mismatch samples anti…+ syn/+ syn…anti, anti…anti & + syn…+ syn glycosyl conformations. The concomitant spontaneous transitions are: a variety of hydrogen bonding patterns, stacking and minor or major groove extrahelical movements (with and without the engagement of hydrogen bonds) involving the mismatch adenines. These transitions frequently happen in anti…anti conformational region compared with the other three regions as revealed from the lifetime of these states. Further, 2D-NOESY experiments indicate that the number of cross-peaks diminishes with the increasing number of A…A mismatches implicating its dynamic nature. The spontaneous extrahelical movement seen in A…A mismatch may be a key pre-trapping event in the mismatch repair due to the accessibility of the base(s) to the sophisticated mismatch repair machinery.

We recently reported the antisense properties of a DNA/RNA heteroduplex oligonucleotide consisting of a phosphorothioate DNA-gapmer antisense oligonucleotide (ASO) strand and its complementary phosphodiester RNA/phosphorothioate 2'-O-methyl RNA strand. When α-tocopherol was conjugated with the complementary strand, the heteroduplex oligonucleotide silenced the target RNA more efficiently in vivo than did the parent single-stranded ASO. In this study, we designed a new type of the heteroduplex oligonucleotide, in which the RNA portion of the complementary strand was replaced with phosphodiester DNA, yielding an ASO/DNA double-stranded structure. The ASO/DNA heteroduplex oligonucleotide showed similar activity and liver accumulation as did the original ASO/RNA design. Structure-activity relationship studies of the complementary DNA showed that optimal increases in the potency and the accumulation were seen when the flanks of the phosphodiester DNA complement were protected using 2'-O-methyl RNA and phosphorothioate modifications. Furthermore, evaluation of the degradation kinetics of the complementary strands revealed that the DNA-complementary strand as well as the RNA strand were completely cleaved in vivo. Our results expand the repertoire of chemical modifications that can be used with the heteroduplex oligonucleotide technology, providing greater design flexibility for future therapeutic applications., Competing Interests: Declaration of Interests T.Y. collaborates with Daiichi Sankyo Company, Ltd; Mitsubishi Tanabe Pharma Corporation; Ono Pharmaceutical Company, Ltd; Rena Therapeutics, Inc.; Takeda Pharmaceutical Company, Ltd; Nanocarrier Pharmaceutical Company, Ltd; and Toray Industries, Inc., and serves as an academic adviser for Rena Therapeutics, Inc. The remaining authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Cellular gene delivery via polycations has wide implications for the potential of gene therapy, but it has remained a challenge due to the plethora of pre- and post-uptake barriers that must be overcome to reach desired efficiency. Herein we report poly(hexamethylene biguanide) (PHMB) as a nano-vector for intracellular delivery of plasmid DNA (pDNA) and oligodeoxynucleotides (ODNs). PHMB and pDNA or ODNs self-assembled into complex nanoparticles at different pH values (7.4 and 12). Their size, charge, cellular uptake, and gene-expression efficiency are assessed and compared to PEI analogues. The systematic results show that the nanoparticles are effective in delivering plasmid DNA and ODNs to model cell lines in culture (HepG2, HEK293T, HeLa), with measurable changes in gene expression levels, comparable to and, in some conditions, even higher than PEI. The well-accepted safety profile of PHMB makes it a valuable candidate for consideration as an effective intracellular DNA vector for further study and potential clinical translation., (Crown Copyright © 2020. Published by Elsevier B.V. All rights reserved.)

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas gene editing systems have enabled molecular geneticists to manipulate prokaryotic and eukaryotic genomes with greater efficiency and precision. CRISPR/Cas provides adaptive immunity in bacterial cells by degrading invading viral genomes. By democratizing this activity into human cells, it is possible to knock out specific genes to disable their function and repair errors. The latter of these activities requires the participation of a single-stranded donor DNA template that provides the genetic information to execute correction in a process referred to as homology directed repair (HDR). Here, we utilized an established cell-free extract system to determine the influence that the donor DNA template length has on the diversity of products from CRISPR-directed gene editing. This model system enables us to view all outcomes of this reaction and reveals that donor template length can influence the efficiency of the reaction and the categories of error-prone products that accompany it. A careful measurement of the products revealed a category of error-prone events that contained the corrected template along with insertions and deletions (indels). Our data provides foundational information for those whose aim is to translate CRISPR/Cas from bench to bedside.

Human apurinic/apyrimidinic endonuclease 1 (APE1) is known to be a critical player of the base excision repair (BER) pathway. In general, BER involves consecutive actions of DNA glycosylases, AP endonucleases, DNA polymerases, and DNA ligases. It is known that these proteins interact with APE1 either at upstream or downstream steps of BER. Therefore, we may propose that even a minor disturbance of protein-protein interactions on the DNA template reduces coordination and repair efficiency. Here, the ability of various human DNA repair enzymes (such as DNA glycosylases OGG1, UNG2, and AAG; DNA polymerase Polβ; or accessory proteins XRCC1 and PCNA) to influence the activity of wild-type (WT) APE1 and its seven natural polymorphic variants (R221C, N222H, R237A, G241R, M270T, R274Q, and P311S) was tested. Förster resonance energy transfer-based kinetic analysis of abasic site cleavage in a model DNA substrate was conducted to detect the effects of interacting proteins on the activity of WT APE1 and its single-nucleotide polymorphism (SNP) variants. The results revealed that WT APE1 activity was stimulated by almost all tested DNA repair proteins. For the SNP variants, the matters were more complicated. Analysis of two SNP variants, R237A and G241R, suggested that a positive charge in this area of the APE1 surface impairs the protein-protein interactions. In contrast, variant R221C (where the affected residue is located near the DNA-binding site) showed permanently lower activation relative to WT APE1, whereas neighboring SNP N222H did not cause a noticeable difference as compared to WT APE1. Buried substitution P311S had an inconsistent effect, whereas each substitution at the DNA-binding site, M270T and R274Q, resulted in the lowest stimulation by BER proteins. Protein-protein molecular docking was performed between repair proteins to identify amino acid residues involved in their interactions. The data uncovered differences in the effects of BER proteins on APE1, indicating an important role of protein-protein interactions in the coordination of the repair pathway.

Low sensitivity of cancer stem cells toward regular cancer therapy strategies is an important issue in the field of cancer remedy. The concept of cancer stem cell elimination has been a topic of interest in the field of molecular medicine for a long time. At the current study, it was aimed to elevate the sensitivity of cancer stem-like cells toward radiotherapy by treating with Oct4-Sox2 complex decoy oligodeoxynucleotides (ODNs). After treating HT29 and HT29-ShE cells with Oct4-Sox2 complex decoy ODNs, and analyzing the cellular uptake and localization of decoys, treated cells and control groups were subjected to irradiation by fractionated 6MV X-ray with a final dose of 2 Gy. Thereafter, the influence of radiotherapy on ODNs treated groups and control group was investigated on cell viability, cell cycle, apoptosis, colonosphere formation and scratch assay. Cellular uptake and localization assays demonstrated that decoy ODNs can efficiently be transfected to the cells and reside in subcellular compartment, where they pose their action on gene regulation. Post radiotherapy analysis indicated statistical significance in decoy ODNs treated cells by means of lower cell viability, cell cycle arrest in G2/M phase, increased cellular apoptosis, and reduced cell motility. Also, formed colonospheres were smaller in size and fewer in numbers. Considering the role of Oct4, and Sox2 transcription factors in signaling pathways of preserving stemness and inducing reverse EMT, application of decoy strategy could increase the sensitivity of cancer cells toward irradiation, which has a potential to eliminate the cancerous cells from tumors and support cancer treatment.

A novel electrochemical nanobiosensor for the detection of miR-155 (as breast cancer biomarker) is introduced . Fe 3 O 4 NPs@Ag core-shell nanoparticles were synthesized and their shape and characteristics were confirmed by scanning electron microscope (SEM) imaging, Fourier-transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD) methods. Synthesized nanoparticles were applied onto the magnetic bar carbon paste electrode and then the amine-modified anti-miR-155 (single-stranded probes) was applied on the modified electrode surface and upon hybridization with target miR-155, resveratrol (RSV) was eventually applied as an electrochemical label on the double-strand oligonucleotide. Differential pulse voltammetry (DPV) of the oxidation peak of RSV was assumed as the final signal by sweeping potential from 0 to 0.6 V (vs. Ag/AgCl). The fabrication process was optimized through a series of experiments and the optimized process was confirmed using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The linear range of the fabricated nanobiosensor was 0.5 fM to 1.0 nM and the detection limit was 0.15 fM. The nanobiosensor was able to pass reproducibility and specificity tests using different types of mismatched target sequences.Spiked real samples of human serum were used to confirm that the nanobiosensor enables detection of miR-155 without any significant interferences from other moieties and molecules. Finally, the molecular dynamics simulation of the RSV interaction with single- and double-stranded oligonucleotide was performed and confirmed the preferential binding of RSV to double-stranded DNA; therefore, it can be used as the electrochemical label of DNA and/or miRNA hybridization-based biosensors. Graphical abstract.

G-triplex (G3) has been recognized as a popular intermediate during the folding of G-quadruplex (G4). This has raised interest to anticipate the ultimate formation of G3 by shortening the G4-forming oligonucleotides with the remaining three G-tracts. Some G3 structures have been validated and their stability has been found to be affected by the loop sequences similar to G4s. In this work, however, we first found that an intermolecular parallel G4 structure was preferred in K+ for the oligonucleotide 5'-TGGGTAGGGCGGG-3' (DZ3) containing only three G-tracts. We screened auramine O (AO) as the appropriate fluorophore with a molecular rotor feature to target this G4 structure. AO bound with DZ3 in a 1 : 4 ratio, as confirmed by isothermal titration calorimetry experiments, suggesting the formation of a tetramolecular G4 structure (4erG4). The excimer emission from the labelled pyrene and the DNA melting behavior at various pHs in the presence of Ag+ proved the formation of the 4erG4 structure rather than the prevalent intramolecular G3 folding. This work demonstrates that one should be cautious while putatively predicting a G3 structure from an oligonucleotide containing three G-tracts.

Dengue is one of the most widespread vector-borne viral diseases in the world. However, the size, heterogeneity, and temporal dynamics of the cell-associated viral reservoir during acute dengue virus (DENV) infection remains unclear. In this study, we analyzed cells infected in vitro with DENV and PBMC from an individual experiencing a natural DENV infection utilizing 5' capture single cell RNA sequencing (scRNAseq). Both positive- and negative-sense DENV RNA was detected in reactions containing either an oligo(dT) primer alone, or in reactions supplemented with a DENV-specific primer. The addition of a DENV-specific primer did not increase the total amount of DENV RNA captured or the fraction of cells identified as containing DENV RNA. However, inclusion of a DENV-specific cDNA primer did increase the viral genome coverage immediately 5' to the primer binding site. Furthermore, while the majority of intracellular DENV sequence captured in this analysis mapped to the 5' end of the viral genome, distinct patterns of enhanced coverage within the DENV polyprotein coding region were observed. The 5' capture scRNAseq analysis of PBMC not only recapitulated previously published reports by detecting virally infected memory and naïve B cells, but also identified cell-associated genomic variants not observed in contemporaneous serum samples. These results demonstrate that oligo(dT) primed 5' capture scRNAseq can detect DENV RNA and quantify virus-infected cells in physiologically relevant conditions, and provides insight into viral sequence variability within infected cells.

Melanomas represent the deadliest form of skin cancers. Due to the intricacy of tumorigenesis, it is emergent to find effective therapies for melanomas. Researches have proved that pimozide inhibits the growth of melanoma, but the limited curing effect needs to be further improved. Nowadays, tumor immunotherapy has been widely recognized as the sole therapy that can eradicate cancers. Cytosine-phosphate-guanine oligodeoxynucleotide (CpG ODN), TLR9 receptor agonist, can significantly enhance anti-tumor immune responses. This study explored the therapeutic effect of pimozide combined with CpG ODN on melanoma-bearing mice. The results showed that pimozide combined with CpG ODN effectively inhibited the growth of melanoma and prolonged the survival of melanoma-bearing mice, inhibited the expression of MMP2 and p-Stat5, increased the infiltration of CD4 + and CD8 + T cells in tumor, raised the ratios of CD4 + , CD8 + T cells and NK cells. These all indicated that the combination treatment improved the anti-tumor effect of pimozide on mice. The anti-tumor mechanism might be attributed to cell apoptosis induction, invasion inhibition, and immune regulation. A more effective combination treatment concerning with pimozide is being under investigation., Competing Interests: Declaration of Competing Interest The authors declared that there is no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Immunotherapy using checkpoint blockade has revolutionized cancer treatment, improving patient survival and quality of life. Nevertheless, the clinical outcomes of such immunotherapy are highly heterogeneous between patients. Depending on the cancer type, the patient response rates to this immunotherapy are limited to 20-30%. Based on the mechanism underlying the antitumor immune response, new therapeutic strategies have been designed with the aim of increasing the effectiveness and specificity of the antitumor immune response elicited by checkpoint blockade agents. The activation of toll-like receptor 9 (TLR9) by its synthetic agonists induces the antitumor response within the innate immunity arm, generating adjuvant effects and priming the adaptive immune response elicited by checkpoint blockade during the effector phase of tumor-cell killing. This review first describes the underlying mechanisms of action and current status of monotherapy using TLR9 agonists and immune checkpoint inhibitors for cancer immunotherapy. The rationale for combining these two agents is discussed, and evidence indicating the current status of such combination therapy as a novel cancer treatment strategy is presented., (Copyright © 2020 Chuang, Tseng, Huang, Huang, Huang and Chuang.)

CLEC12A has been proposed as a suitable target for delivering antigen to dendritic cells (DCs) to enhance vaccine efficacy both in human and mouse. In this study, we have characterized the porcine homolog of CLEC12A (poCLEC12A). Using new monoclonal antibodies (mAb), raised against its ectodomain, poCLEC12A was found to be expressed on alveolar macrophages, blood conventional type 1 and type 2 DCs and plasmacytoid DCs, but not on monocytes, T cells, B cells or NK cells, in contrast to its human and murine homologs. Western blot analysis showed that in alveolar macrophages this receptor is expressed both as a monomer and a dimer. After binding to DCs, anti- poCLEC12A mAb was efficiently internalized. No significant changes were observed in TNFα or IFNα secretion by plasmacytoid DCs stimulated with either CpGs (ODN2216) or polyinosinic-polycytidylic acid (poly I:C), upon incubation with mAb. These results provide the basis for future investigations aimed to assess the ability of anti-poCLEC12A mAbs to improve vaccine efficacy by targeting antigen to DCs., (Copyright © 2020 Álvarez, Nieto-Pelegrín, Martínez de la Riva, Toki, Poderoso, Revilla, Uenishi, Ezquerra and Domínguez.)

Different mutations of the OTOF gene, encoding for otoferlin protein expressed in the cochlear inner hair cells, induces a form of deafness that is the major cause of nonsyndromic recessive auditory neuropathy spectrum disorder in humans. We report the generation of the first large animal model of OTOF mutations using the CRISPR system associated with different Cas9 components (mRNA or protein) assisted by single strand oligodeoxynucleotides (ssODN) to induce homology-directed repair (HDR). Zygote microinjection was performed with two sgRNA targeting exon 5 and 6 associated to Cas9 mRNA or protein (RNP) at different concentrations in a mix with an ssODN template targeting HDR in exon 5 containing two STOP sequences. A total of 73 lambs were born, 13 showing indel mutations (17.8%), 8 of which (61.5%) had knock-in mutations by HDR. Higher concentrations of Cas9-RNP induced targeted mutations more effectively, but negatively affected embryo survival and pregnancy rate. This study reports by the first time the generation of OTOF disrupted sheep, which may allow better understanding and development of new therapies for human deafness related to genetic disorders. These results support the use of CRISPR/Cas system assisted by ssODN as an effective tool for gene editing in livestock.

Branched oligonucleotides containing a biologically relevant DNA lesion, dCyd341, which involves an interstrand crosslink between a cytosine base on one strand and a ribose moiety on the opposite strand, were prepared in a single automated solid-phase synthesis. For this, we first prepared the phosphoramidite analogue of dCyd341 bearing an orthogonal levulinyl protecting group. Then, following the synthesis of the first DNA strand containing dCyd341, the levulinic group was removed and the synthesis was then continued from the free base hydroxyl group at the branching point, using traditional phosphoramidites. The synthesized oligonucleotides were fully characterized by MALDI-TOF/MS and were enzymatically digested, and the presence of the lesion was confirmed by HPLC-MS/MS and the sequence was finally controlled upon exonuclease digestion followed by MALDI-TOF/MS analysis. The developed strategy was successfully employed for the preparation of several short linear and branched oligonucleotides containing the aforementioned lesion.

6-Nitrochrysene (6-NC), the most potent carcinogen evaluated by the newborn mouse assay, is metabolically activated by nitroreduction and a combination of ring oxidation and nitroreduction pathways. The nitroreduction pathway yields three major DNA adducts: at the C8 and N 2 positions of 2'-deoxyguanosine (dG), N -(dG-8-yl)-6-AC and 5-(dG- N 2 -yl)-6-AC, and at the C8 position of 2'-deoxyadenosine (dA), N -(dA-8-yl)-6-AC. A nucleotide excision repair assay demonstrated that N -(dA-8-yl)-6-AC is repaired much more slowly than many other bulky DNA adducts, including the other DNA adducts formed by 6-NC. But neither the total synthesis nor evaluation of other biological activities of this dA adduct has ever been reported. Herein, we report a convenient synthesis of the 6-NC-derived dA adduct by employing the Buchwald-Hartwig coupling strategy, which provided a high yield of the protected N -(dA-8-yl)-6-AC. The deprotected nucleoside showed syn conformational preference by NMR spectroscopy. Following DMT protection of the 5'-hydroxyl, N -(dA-8-yl)-6-AC was converted to its 3'-phosphoramidite, which was used to prepare oligonucleotides containing a single N -(dA-8-yl)-6-AC adduct. Circular dichroism spectra of the adducted duplex showed only a slight departure from the B-DNA helix profile of the control duplex. The 15-mer N -(dA-8-yl)-6-AC oligonucleotide was used to construct a single-stranded plasmid vector containing a single adduct, which was replicated in Escherichia coli . Viability of the adducted construct was ∼60% of the control, indicating slower translesion synthesis of the adduct, which increased to nearly 90% upon induction of the SOS functions. Without SOS, the mutation frequency (MF) of the adduct was 5.2%, including 2.9% targeted and 2.3% semi-targeted mutations. With SOS, the targeted MF increased 3-fold to 9.0%, whereas semi-targeted mutation increased only marginally to 3.2%. The major type of targeted mutation was A*→G in both uninduced and SOS-induced cells.

Human neutrophils are the first line of defense against bacterial and viral infections. They eliminate pathogens through phagocytosis, which activate the 5-lipoxygenase (5-LOX) pathway resulting in synthesis of leukotrienes. Using HPLC analysis, flow cytometry, and other biochemical methods, we studied the effect of synthetic oligodeoxyribonucleotides (ODNs) able to fold into G-quadruplex structures on the main functions of neutrophils. Designed ODNs contained four human telomere TTAGGG repeats (G4) including those with phosphorothioate oligoguanosines attached to the end(s) of G-quadruplex core. Just modified analogues of G4 was shown to more actively than parent ODN penetrate into cells, improve phagocytosis of Salmonella typhimurium bacteria, affect 5-LOX activation, the cytosol calcium ion level, and the oxidative status of neutrophils. As evident from CD and UV spectroscopy data, the presence of oligoguanosines flanking G4 sequence leads to dramatic changes in G-quadruplex topology. While G4 folds into a single antiparallel structure, two main folded forms have been identified in solutions of modified ODNs: antiparallel and dominant, more stable parallel. Thus, both the secondary structure of ODNs and their ability to penetrate into the cytoplasm of cells are important for the activation of neutrophil cellular effects. Our results offer new clues for understanding the role of G-quadruplex ligands in regulation of integral cellular processes and for creating the antimicrobial agents of a new generation., Competing Interests: The authors declare no conflict of interest.

Oligodeoxynucleotides containing unmethylated CpG dinucleotides (CpG-ODN) can be specifically recognized by Toll-like receptor 9 (TLR9), provoking innate immune responses. Designed according to this structural feature, many synthetic phosphorothioate CpG-ODNs successfully activate macrophages. However, it is difficult to find potent stimulatory CpG-DNA fragments in microbial genomes. Therefore, whether microbial CpG-DNA substantially contributes to infectious and immune diseases remains controversial. In this study, high-throughput scanning was carried out for thousands of bacterial genomes with bioinformatics tools to comprehensively evaluate the distribution of CpG-DNA fragments. A random sampling test was then performed to verify their immunostimulatory properties by experiments in vitro and in vivo. Natural TLR9-dependent and potent stimulatory CpG-DNA fragments were found in microbial genomes. Interestingly, highly conserved stimulatory CpG-DNA fragments were found in 16S and 23S rDNA sequences with multiple copies, while others were species-specific. Additionally, we found that the reported active motifs were mostly non-stimulatory in natural CpG fragments. This evidence indicates that the previous structural descriptions of functional CpG-ODNs are incomplete. Our study has assessed the distribution of microbial CpG-DNA fragments, and identified natural stimulatory CpG-DNA fragments. These findings provide a deeper understanding of CpG-ODN structures and new evidence for microbial DNA inflammatory function and pathogenicity.

Quantification of plasma membrane proteins (PMPs) is crucial for understanding the fundamentals of cellular signaling systems and their related diseases. In this work, a super-quadruplex scaffold was designed to regulate assembly of oligonucleotide-grafted AIEgens for detection of PMPs. The nonfluorescence oligonucleotide-grafted AIEgen (Oligo-AIEgen) was firstly synthesized by attaching the AIEgen to 3'-terminus of the oligonucleotide through click chemistry. Meanwhile, the tetramolecular hairpin-conjugated super-quadruplex (THP-G4) as cleavage element and signal enhancement scaffold composited of three elements: a substrate sequence of DNAzyme in the loop region, partial hybridization region in the stem, and six guanine nucleotides to form G-quadruplex. Once the DNAzyme was anchored on the specific PMPs through aptamer-protein recognition, the substrate sequence on the loop of THP-G4 was cleaved by DNAzyme with the aid of cofactor Mn II , resulting in the conformation switch of THP-G4 to the activated G-quadruplex scaffold. The latter could assemble Oligo-AIEgens to generate aggregation-induced emission (AIE) enhancement, resulting in a simple and sensitive strategy for detection of membrane proteins. Moreover, the DNAzyme continuously cut the next THP-G4 to achieve recycling amplification. Under the optimized conditions, this AIE-based strategy exhibited good linear relationship with the logarithm of MUC1 concentration from 0.01 to 10 μg mL -1 with the limit of detection down to 4.3 ng mL -1 . The G4-assembled AIEgens provides a universal platform for detecting various biomolecules and a proof-of concept for AIE biosensing., (Copyright © 2019 Elsevier B.V. All rights reserved.)