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 IC50 value of 10.6 nM. In addition, 12a demonstrated high efficacy in various colon cancer cell lines as indicated by GI50 values ranging from 0.24 to 1.26 µM. Notably, 12a inhibited the phosphorylation of Lck in Colo201 cells. Overall, the anti-proliferative effects of 12a on diverse cancer cell lines highlights its potential application for the treatment of various cancer types.

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 (64Cu), with a clinically practical half-life (t1/2 = 12.7 h) and decay properties, was selected as the radioisotope for molecular PET imaging. An anti-epidermal growth factor receptor (anti-EGFR) antibody was used to achieve target-specific delivery. Simultaneously, the chemotherapeutic agent doxorubicin (Dox) was encapsulated within the liposomes using a pH-gradient method. The conjugates of 64Cu-labeled and anti-EGFR antibody-conjugated micelles were inserted into the doxorubicin-encapsulating liposomes via a post-insertion procedure (64Cu-Dox-immunoliposomes). We evaluated the size and zeta-potential of the liposomes and analyzed target-specific cell binding and cytotoxicity in EGFR-positive cell lines. Then, we analyzed the specific therapeutic effect and PET imaging of the 64Cu-Dox-immunoliposomes with the A549 xenograft mouse model. In vivo therapeutic experiments on the mouse models demonstrated that the doxorubicin-containing 64Cu-immunoliposomes effectively inhibited tumor growth. Moreover, the 64Cu-immunoliposomes provided superior in vivo PET images of the tumors compared to the untargeted liposomes. We suggest that nanoparticles will be the potential platform for cancer treatment as a widely applicable theranostic system.

Discovery of thiophen-2-ylmethylene bis-dimedone derivatives as novel WRN inhibitors for treating cancers with microsatellite instability.

Microsatellite instability (MSI) is a hypermutable condition caused by DNA mismatch repair system defects, contributing to the development of various cancer types. Recent research has identified Werner syndrome ATP-dependent helicase (WRN) as a promising synthetic lethal target for MSI cancers. Herein, we report the first discovery of thiophen-2-ylmethylene bis-dimedone derivatives as novel WRN inhibitors for MSI cancer therapy. Initial computational analysis and biological evaluation identified a new scaffold for a WRN inhibitor. Subsequent SAR study led to the discovery of a highly potent WRN inhibitor. Furthermore, we demonstrated that the optimal compound induced DNA damage and apoptotic cell death in MSI cancer cells by inhibiting WRN. This study provides a new pharmacophore for WRN inhibitors, emphasizing their therapeutic potential for MSI cancers.

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. In this study, we devised a CRISPR activation system using a DNA regulator that can be activated by miRNAs. The designed regulator was divided into two parts. The inhibition component consisted of the protospacer-adjacent motif (PAM) and seed sequence, which are important for Cas9 target recognition and bind to the ribonucleoprotein (RNP) complex for inhibition. The miRNA recognition component has a single-stranded toehold DNA for target miRNA binding and a partial double-stranded DNA complementary to the remaining miRNA sequence. In the presence of target miRNAs, the structure of the regulator is disrupted by the miRNAs, leading to its dissociation from the RNP complex and subsequent restoration of CRISPR activity. This method is easy to design and can be applied to various miRNAs via simple sequence manipulation. Therefore, this strategy provides a general platform for controlled genome editing.

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). ExIT uses a newly designed, self-folding primer (SP) with two key functions. Hairpin structures are formed when the extended strand of the SP hybridizes, exposing the priming site for continuous binding of the new SP. This results in exponential amplification with only two primers, unlike conventional LAMP primer systems. Additionally, an unnatural base was introduced into the SP, which terminated the extension of polymerase and generated a ssDNA amplicon. This makes it easier to design and apply probes, reducing the possibility of false-positive results even if non-specific amplicons are produced. Through this strategy, we confirmed a sensitivity of 90 copies (3.6 copies/µL) and verified the specificity by testing for the presence or absence of non-complementary targets. Therefore, the validation of the ExIT was completed. In conclusion, ExIT will be key to solving the complexity of conventional LAMP design and offers great potential for successfully introducing sequence-specific probes to improve false positives.

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 0.001-0.005% VAF; however, it requires expensive equipment and time-consuming protocols. Therefore, various PCR methods for discriminating SNVs have been developed; nonetheless, non-specific amplification cannot be avoided even in the absence of mutations, which hampers the accurate diagnosis of SNVs. In this study, we introduce single-nucleotide variant on-off discrimination-PCR (Soo-PCR), a highly accurate and practical method that uses a 3'-end tailing primer for the on-off discrimination of low-abundance mutant-type targets, including SNVs. Soo-PCR minimizes the chance of incorrect judgments owing to its high discriminating power. Cancer markers, such as KRAS G12D, EGFR L858R, and EGFR T790M mutations, containing 0.1% VAF, were clearly detected in under 2 h with a high reliability comparable with that of ddPCR. This new method serves as a practical approach to accurately detect and evaluate low-abundance mutations in a user-friendly manner.

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. It is found that the nucleobases at positions 7-12 of the 18-nucleotide-long activator are critical to creating bright NCBs and positions 4-6 and 2-4 are hotspots to generate yellow-orange and red POTs, respectively. Based on these findings, a "zipper-bag" model is proposed that can explain how these hotspots facilitate the formation of distinct silver cluster chromophores and alter their chemical yields. Combining high-throughput screening with machine-learning algorithms, a pipeline is established to design bright and multicolor NCBs in silico.

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.

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. The three-step process of a general qPCR was reduced to a two-step process. The annealing/extension temperatures were increased to minimize the differences between the denaturation temperature and the annealing/extension temperatures. Subsequently, the time for each of these steps was reduced and, finally, the denaturation temperature was lowered. Taq polymerase was replaced with SD polymerase because it has strand displacement activity and is efficient in amplifying partial dsDNA at lower denaturation temperatures. In the two-step qPCR of genomic DNA using SD polymerase, the final conditions included an initial denaturation at 92 °C for 2 min, and 1 s at each cycling step with a denaturation temperature of 87 °C and an annealing/extension temperature of 72 °C. Amplification of the nucleocapsid (N) gene of SARS-CoV-2 RNA virus was evaluated at a template concentration as low as 10 copies. This method, named SF-qPCR (strand displacement-based fast quantitative polymerase chain reaction), can stably detect less than 10 copies of DNA and RNA within 25-40 min. This new protocol allows for sensitive and rapid detection of important DNA and RNA targets in clinical diagnosis. Supplementary Information: The online version contains supplementary material available at 10.1007/s13206-021-00044-x.

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). Interaction between the loop part of HP and its target exposes a single strand of nucleotides, which acts as the template for PPR. A pair of protease and zymogen-conjugated nucleic acids associates with the adjacent regions of the template, resulting in an enhanced proteolysis reaction between protease and zymogen. The activated zymogen then generates a color signal through the hydrolysis of a chromogenic substrate. The combination of HP converter and PPR allowed the same pair of protease- and zymogen-nucleic acids to be used for different RNAs. Guidelines were provided for designing HP converters based on computational analyses and experimental characterizations. This strategy using an HP converter and PPR has been successfully applied to develop simple isothermal methods for the detection of various RNAs, including several microRNAs and KRAS mRNA, in the picomolar range in 1 h. The simplicity of designing HP converters and the beneficial properties of PPR as POC tests would enable the development of novel methods to detect RNAs under low-resource conditions.

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. The diffusion coefficient of lipid probes, as well as the type of diffusion determined by an exponent α, which is the slope of the log-log plot of mean squared displacement as a function of time lag, were analyzed. We found that the number of molecules with a lower diffusion coefficient increased as cells became senescent. The changes in the population with a lower diffusion coefficient, observed after methyl-ß-cyclodextrin treatment, and the increase in ceramide levels, detected using a ceramide-specific antibody, suggest that ceramide-rich lipid rafts were enhanced in senescent cells. Our results emphasize the importance of membrane properties in cellular senescence and might serve as a base for in-depth studies to determine how such domains facilitate the signaling pathway specific to cellular senescence.

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. Furthermore, KAS-08 was validated as an effective STING pathway activator in vitro and in vivo. The synergistic effect of cGAMP-mediated immunity and efficient anti-cancer effects successfully demonstrated the therapeutic potential of KAS-08 for combination therapy in cancer treatment.

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. Combining cleavage rates and binding specificities on the same target libraries, we benchmarked five SpCas9 variants and AsCas12a. A biophysical model built from these data sets revealed mechanistic insights into off-target cleavage. Engineered Cas9s, especially Cas9-HF1, dramatically increased cleavage specificity but not binding specificity compared to wtCas9. Surprisingly, AsCas12a cleavage specificity differed little from that of wtCas9. Initial DNA cleavage sites and end trimming varied by nuclease, guide RNA and the positions of mispaired nucleotides. More broadly, NucleaSeq enables rapid, quantitative and systematic comparisons of specificity and cleavage outcomes across engineered and natural nucleases.

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. However, it tolerates mismatches in regions distant from the PAM sequence (TTTN or TTN) in the protospacer, and off-target cleavage issues may become more problematic when Cas12a activity is improved for therapeutic purposes. Therefore, we investigated off-target cleavage by Cas12a and modified the Cas12a (cr)RNA to address the off-target cleavage issue. We developed a CRISPR-Cas12a that can induce mutations in target DNA sequences in a highly specific and effective manner by partially substituting the (cr)RNA with DNA to change the energy potential of base pairing to the target DNA. A model to explain how chimeric (cr)RNA guided CRISPR-Cas12a and SpCas9 nickase effectively work in the intracellular genome is suggested. Chimeric guide-based CRISPR- Cas12a genome editing with reduced off-target cleavage, and the resultant, increased safety has potential for therapeutic applications in incurable diseases caused by genetic mutations.

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. The starting and stopping of the walker, and its association and dissociation from the microparticles, could also be dynamically controlled by pH. The simple, programmable, and robust nature of this proton-controlled walker now provides the impetus for the development of a wide variety of more practical nanomachines.

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. This ligation-based miRNA assay is highly specific for mature miRNAs, discriminating among related miRNAs that differ by as little as one nucleotide. It exhibits a dynamic range of seven orders of magnitude with a detection limit of 500 aM, and could be also used for the quantification of other small RNA molecules such as short interfering RNAs (siRNAs).

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. The platinum-aptamer probe contained several guanine (G) bases bound to the 3,4,5-trimethoxyphenyl-glyoxal (TMPG) donor group at the 5' end, a fluorescent acceptor (6-carboxy-2',4,7,7'-tetrachlorofluorescein, TET) at the 3' end, and a streptavidin aptamer sequence in which several base pairs were replaced by the G-G mismatch to induce the platinum-oligonucleotide coordination. The chemiluminescence (CL) generated by TMPG/G bases is transferred to the acceptor (TET). In the presence of platinum, the platinum-aptamer probe was folded such that the G bases at the 5' end and TET at the 3' were in close proximity. The complex was separated using streptavidin-coated magnetic beads by the addition of TMPG to form the TMPG/G bases complex. The ultraweak CL from the TMPG/G bases was strongly enhanced by TET. This novel CRET-based method can be easily performed with high limit of detection (50 ng·mL-1) and selectivity over other metal ions. This technique provides a novel method for simple, fast, and convenient point-of-care diagnostics for monitoring proteins and metal ions. Graphical abstract Schematic presentation of chemiluminescence resonance energy transfer (CRET) detection of platinum(II) by Pt-base pair coordination to the aptamer. TMPG: 3,4,5-trimethoxyphenyl-glyoxal, fluorophore TET: 6-carboxy-2',4,7,7'-tetrachlorofluorescein.

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). We show that supercharged variants of green fluorescent protein can assemble into a variety of architectures including a well-defined symmetrical 16-mer structure that we solved using cryo-electron microscopy at 3.47 Å resolution. The 16-mer is composed of two stacked rings of octamers, in which the octamers contain supercharged proteins of alternating charges, and interactions within and between the rings are mediated by a variety of specific electrostatic contacts. The ready assembly of this structure suggests that combining oppositely supercharged pairs of protein variants may provide broad opportunities for generating novel architectures via otherwise unprogrammed interactions.

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. Because of the helper template, a slight change in platinum concentration would significantly change the signal in the qPCR. This novel helper qPCR technique easily detects platinum with high sensitivity (1 ng/mL) and selectivity over other metal ions. Therefore, it will be a promising technique for the detection of platinum in biomedical and environmental samples.

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). Upon the addition of deoxynucleoside triphosphates and a polymerase, concatemers quickly formed, and those random sequences that templated the insertion of purines, especially during initiation, quickly predominated. Over several serial transfers, particular sequences accumulated, and in isolation these were shown to outcompete less efficient replicators.