Development of a TBK1 and ALK dual inhibitor for alleviating depressive behavior via anti-inflammatory effects.

Polypharmacology offers innovative strategies for treating immune and inflammatory dysregulation in complex diseases. Here, we identified ALS-04, a dual inhibitor of TANK-binding kinase 1 (TBK1) and anaplastic lymphoma kinase (ALK), which are closely linked to stimulator of interferon genes (STING)-mediated immune responses. ALS-04 effectively suppressed 2'3'-cyclic GMP-AMP (cGAMP)- and lipopolysaccharide (LPS)-induced type I interferon and pro-inflammatory responses by targeting the STING-TBK1 and STING-ALK pathways.

Development of PET Radioisotope Copper-64-Labeled Theranostic Immunoliposomes for EGFR Overexpressing Cancer-Targeted Therapy and Imaging.

Combining standard surgical procedures with personalized chemotherapy and the continuous monitoring of cancer progression is necessary for effective NSCLC treatment. In this study, we developed liposomal nanoparticles as theranostic agents capable of simultaneous therapy for and imaging of target cancer cells. Copper-64 (Cu), with a clinically practical half-life ( = 12.

Identification of 3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one scaffolds as potent Lck inhibitors as anti-cancer agents.

Lymphocyte-specific protein tyrosine kinase (Lck) plays vital roles in the T-cell receptor- mediated development, function, and differentiation of T-cells. Given its substantial involvement in T cell signaling, irregularities in the expression and functionality of Lck may lead to various diseases, including cancer. In this study, we found that compound 12a exerted significant inhibitory potency against Lck with an IC value of 10.

MiRNA-Responsive CRISPR-Cas System via a DNA Regulator.

Clustered regularly interspaced short palindromic repeats (CRISPR)- CRISPR-associated protein 9 (Cas9) genome editing technology is widely used for gene editing because it provides versatility in genetic manipulation. Several methods for regulating CRISPR activity already exist for accurate editing, but these require complex engineering. Thus, a simple and convenient regulatory system is required.

Development of extension-mediated self-folding isothermal amplification technology for SARS-CoV-2 diagnosis.

The coronavirus disease (COVID-19) pandemic has highlighted the importance of rapid and accurate diagnosis, and loop-mediated isothermal amplification (LAMP) has become a popular method because of its powerful amplification ability using a simple instrument such as a heater or water bath. However, LAMP has limitations such as the complexity of primer design and the difficulty of designing sequence-specific probes, leading to non-specific amplicons and false-positive results. To overcome these limitations, we developed a novel isothermal amplification system called the Extension-mediated self-folding Isothermal amplification Technology (ExIT).

Improving the Accuracy of Single-Nucleotide Variant Diagnosis Using On-Off Discriminating Primers.

Early detection of rare mutations through liquid biopsy can provide real-time information related to cancer diagnosis, prognosis, and treatment outcomes. Cell-free DNA samples used in liquid biopsies contain single-nucleotide variants (SNVs) with a variant allele frequency (VAF) of approximately ≤1%. Droplet digital polymerase chain reaction (ddPCR) is considered the gold standard of sequencing using liquid samples, generating amplicons from samples containing mutations with 0.

Massively Parallel Selection of NanoCluster Beacons.

NanoCluster Beacons (NCBs) are multicolor silver nanocluster probes whose fluorescence can be activated or tuned by a proximal DNA strand called the activator. While a single-nucleotide difference in a pair of activators can lead to drastically different activation outcomes, termed polar opposite twins (POTs), it is difficult to discover new POT-NCBs using the conventional low-throughput characterization approaches. Here, a high-throughput selection method is reported that takes advantage of repurposed next-generation-sequencing chips to screen the activation fluorescence of ≈40 000 activator sequences.

Expansion of the prime editing modality with Cas9 from Francisella novicida.

Prime editing can induce a desired base substitution, insertion, or deletion in a target gene using reverse transcriptase after nick formation by CRISPR nickase. In this study, we develop a technology that can be used to insert or replace external bases in the target DNA sequence by linking reverse transcriptase to the Francisella novicida Cas9, which is a CRISPR-Cas9 ortholog. Using FnCas9(H969A) nickase, the targeting limitation of existing Streptococcus pyogenes Cas9 nickase [SpCas9(H840A)]-based prime editing is dramatically extended, and accurate prime editing is induced specifically for the target genes in human cell lines.

SF-qPCR: Strand Displacement-Based Fast Quantitative Polymerase Chain Reaction.

Unlabelled: Nucleic acid testing (NAT) is important for the identification and quantification of specific nucleic acid targets, both DNA and RNA, in life sciences and clinical diagnostics. Nucleic acid amplification can be a time-consuming step in NAT using the polymerase chain reaction (PCR) assay. Therefore, this study aimed to develop a simple method to reduce the amplification time while maintaining the PCR system.

Development of Small-Molecule STING Activators for Cancer Immunotherapy.

In cancer immunotherapy, the cyclic GMP-AMP synthase-stimulator of interferon genes (STING) pathway is an attractive target for switching the tumor immunophenotype from 'cold' to 'hot' through the activation of the type I interferon response. To develop a new chemical entity for STING activator to improve cyclic GMP-AMP (cGAMP)-induced innate immune response, we identified KAS-08 via the structural modification of DW2282, which was previously reported as an anti-cancer agent with an unknown mechanism. Further investigation revealed that direct STING binding or the enhanced phosphorylation of STING and downstream effectors were responsible for DW2282-or KAS-08-mediated STING activity.

Development of one-step isothermal methods to detect RNAs using hairpin-loop signal converters and proximity proteolysis reaction.

Ribonucleic acids (RNAs) provide valuable information for biological systems and act as important indicators of disease states. RNAs are diverse in size and structure, and various strategies have been proposed for the detection of nucleic acids; however, developing them into point-of-care (POC) tests has been challenging as most of them consist of complex time-consuming steps. Here, we propose a strategy to assay RNAs using a hairpin-loop (HP) converter and proximity proteolysis reaction (PPR).

Probing Physical Properties of the Cellular Membrane in Senescent Cells by Fluorescence Imaging.

Cellular senescence is the irreversible cell cycle arrest in response to various types of stress. Although the plasma membrane and its composition are significantly affected by cellular senescence, detailed studies on the physical properties of the plasma membrane have shown inconclusive results. In this study, we utilized both ensemble and single-molecule fluorescence imaging to investigate how membrane properties, such as fluidity, hydrophobicity, and ganglioside GM1 level are affected by cellular senescence.

Massively parallel kinetic profiling of natural and engineered CRISPR nucleases.

Engineered SpCas9s and AsCas12a cleave fewer off-target genomic sites than wild-type (wt) Cas9. However, understanding their fidelity, mechanisms and cleavage outcomes requires systematic profiling across mispaired target DNAs. Here we describe NucleaSeq-nuclease digestion and deep sequencing-a massively parallel platform that measures the cleavage kinetics and time-resolved cleavage products for over 10,000 targets containing mismatches, insertions and deletions relative to the guide RNA.

Enhancement of target specificity of CRISPR-Cas12a by using a chimeric DNA-RNA guide.

The CRISPR-Cas9 system is widely used for target-specific genome engineering. CRISPR-Cas12a (Cpf1) is one of the CRISPR effectors that controls target genes by recognizing thymine-rich protospacer adjacent motif (PAM) sequences. Cas12a has a higher sensitivity to mismatches in the guide RNA than does Cas9; therefore, off-target sequence recognition and cleavage are lower.

Dynamic Programming of a DNA Walker Controlled by Protons.

We have now constructed a four-legged DNA walker based on toehold exchange reactions whose movement is controlled by alternating pH changes. A well-characterized, pH-responsive CG-C triplex DNA was embedded into a tetrameric catalytic hairpin assembly (CHA) walker. The proton-controlled walker could autonomously move on otherwise unprogrammed microparticles surface, and the walking rate and steps of walking were efficiently controlled by pH.

Ultrasensitive detection of miRNA via one-step rolling circle-quantitative PCR (RC-qPCR).

A novel microRNA (miRNA) quantification method has been developed using one-step rolling circle-quantitative PCR (RC-qPCR) analysis. Vent (exo-) DNA polymerase is firstly utilized to combine a rolling circle amplification (RCA) and qPCR in one step with high sensitivity and specificity in our RC-qPCR assay. Before performing the RC-qPCR, a padlock probe is ligated only when it is perfectly hybridized with miRNA.

A chemiluminescence resonance energy transfer strategy and its application for detection of platinum ions and cisplatin.

A novel chemiluminescence resonance energy transfer (CRET) system was developed and combined with a structure-switching aptamer for the highly sensitive detection of platinum. Platinum was chosen as a model analyte to demonstrate the generality of the new CRET system. This aptameric platform consisted of a streptavidin labeled aptamer against platinum and a streptavidin-coated magnetic bead for the selective separation of platinum-bound aptamer.

Selection of self-priming molecular replicators.

Self-priming amplification of oligonucleotides is possible based on foldback of 3' ends, self-priming, and concatemerization, especially in the presence of phosphorothioate linkages. Such a simple replicative mechanism may have led to the accumulation of specific replicators at or near the origin of life. To determine how early replicators may have competed with one another, we have carried out selections with phosphorothiolated hairpins appended to a short random sequence library (N10).

A novel helper qPCR system for platinum detection via Pt-DNA coordination.

A novel real-time polymerase chain reaction (qPCR) platform for the simple and robust detection of platinum is described for the first time. Compared with conventional qPCR, a helper template, which is related to the active template for performing qPCR, was introduced in our helper qPCR system. Several guanine (G) bases were introduced in the helper template to obtain a platinum-responsive on/off switch based on G-Pt-G coordination.

Supercharging enables organized assembly of synthetic biomolecules.

Symmetrical protein oligomers are ubiquitous in biological systems and perform key structural and regulatory functions. However, there are few methods for constructing such oligomers. Here we have engineered completely synthetic, symmetrical oligomers by combining pairs of oppositely supercharged variants of a normally monomeric model protein through a strategy we term 'supercharged protein assembly' (SuPrA).

Universally applicable, quantitative PCR method utilizing fluorescent nucleobase analogs.

We herein describe a novel quantitative PCR (qPCR) method, which operates in both signal-off and on manners, by utilizing a unique property of fluorescent nucleobase analogs. The first, signal-off method is developed by designing the primers to contain pyrrolo-dC (PdC), one of the most common fluorescent nucleobase analogs. The specially designed single-stranded primer is extended to form double-stranded DNA during PCR and the fluorescence signal from the PdCs incorporated in the primer is accordingly reduced due to its conformation-dependent fluorescence properties.

Phosphorothioated Primers Lead to Loop-Mediated Isothermal Amplification at Low Temperatures.

Loop-mediated isothermal amplification (LAMP) is an extremely powerful tool for the detection of nucleic acids with high sensitivity and specificity. However, LAMP shows optimal performance at around 65 °C, which limits applications in point-of-care-testing (POCT). Here, we have developed a version of LAMP that uses phosphorothioated primers (PS-LAMP) to enable more efficient hairpin formation and extension at the termini of growing concatamers, and that therefore works at much lower temperatures.

A Simple, Cleated DNA Walker That Hangs on to Surfaces.

We designed and demonstrated a single-legged or unipedal walker that has a "cleat" that allows it to persistently associate with a track and make autonomous decisions about movement. The walker is highly processive over long periods of time, as shown by its movement over a microparticle surface suffused with substrate. The simple design can be readily optimized on the basis of simple energetic considerations.