RESUMO
Targeting "undruggable" targets with intrinsically disordered structures is of great significance for the treatment of disease. The transcription factor c-Myc controls global gene expression and is an attractive therapeutic target for multiple types of cancers. However, due to the lack of defined ligand binding pockets, targeted c-Myc have thus far been unsuccessful. Herein, to address the dilemma of lacking ligands, an efficient and high throughput aptamer screening strategy is established, named polystyrene microwell plate-based systematic evolution of ligands by exponential enrichment (microwell-SELEX), and identify the specific aptamer (MA9C1) against c-Myc. The multifunctional aptamer-based Proteolysis Targeting Chimeras (PROTAC) for proteolysis of the c-Myc (ProMyc) is developed using the aptamer MA9C1 as the ligand. ProMyc not only significantly degrades c-Myc by the ubiquitin-proteasome system, but also reduces the Max protein, synergistically inhibiting c-Myc transcriptional activity. Combination of the artificial cyclization and anti-PD-L1 aptamer (PA1)-based delivery system, circular PA1-ProMyc chimeras achieve tumor regression in the xenograft tumor model, laying a solid foundation for the development of efficacious c-Myc degrader for the clinic. Therefore, this aptamer-based degrader provides an invaluable potential degrader in drug discovery and anti-tumor therapy, offering a promising degrader to overcome the challenge of targeting intractable targets.
Assuntos
Aptâmeros de Nucleotídeos , Proteólise , Proteínas Proto-Oncogênicas c-myc , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Humanos , Aptâmeros de Nucleotídeos/farmacologia , Animais , Camundongos , Proteólise/efeitos dos fármacos , Linhagem Celular Tumoral , Modelos Animais de Doenças , Técnica de Seleção de Aptâmeros/métodos , Antineoplásicos/farmacologiaRESUMO
Checkpoint inhibitor antibody therapy by blocking the interaction of surface programmed death-ligand 1(PD-L1) and programmed cell death protein 1(PD-1) has promising advantages in cancer immunotherapy. However, the response of many patients remains unsatisfactorily, suspected to be relevant to PD-L1 located in other cellular compartments and antibodies do not have access to the intracellular compartments. Herein, we identify a PD-L1-targeting DNA aptamer (PA9-1) with dual roles, including an antagonist and a delivery agent dependent on PD-L1 internalization. And we design the PD-L1-targeting antagonistic aptamer-ASO delivery system (PA9-1-ASO), with synergistic inhibitory PD-L1 activity involving the combination of blockade and silencing mechanisms. This chimera not only blocks PD-L1/PD-1 but also achieves targeted delivery of the conjugated ASO to reduce both surface PD-L1 and total PD-L1 expression. Compared with the single blockade, this chimera with the dual inhibitory function synergistically inhibits PD-L1 to amplify immunotherapeutic efficacy, providing a promising synergistic strategy for immunotherapy.
Assuntos
Antígeno B7-H1 , Neoplasias , Humanos , Antígeno B7-H1/uso terapêutico , Receptor de Morte Celular Programada 1 , Neoplasias/tratamento farmacológico , ImunoterapiaRESUMO
Inhibition of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and excessive inflammation is the current task in the prevention and treatment of corona vireus disease 2019 (COVID-19). Here, a dual-function circular aptamer-ASO chimera (circSApt-NASO) is designed to suppress SARS-CoV-2 replication and inflammation. The chemically unmodified circSApt-NASO exhibits high serum stability by artificial cyclization. It is also demonstrated that the SApt binding to spike protein enables the chimera to be efficiently delivered into the host cells expressing ACE2 along with the infection of SARS-CoV-2. Among them, the SApt potently inhibits spike-induced inflammation. The NASO targeting to silence N genes not only display robust anti-N-induced inflammatory activity, but also achieve efficient inhibition of SARS-CoV-2 replication. Overall, benefiting from the high stability of the cyclization, antispike aptamer-dependent, and viral infection-mediate targeted delivery, the circSApt-NASO displays robust potential against authentic SARS-CoV-2 and Omicron, providing a promising specific anti-inflammatory and antiproliferative reagent for therapeutic COVID-19.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Inflamação , Proliferação de CélulasRESUMO
Understanding how long noncoding RNAs (lncRNAs) cooperate with splicing factors (SFs) in alternative splicing (AS) control is fundamental to human biology and disease. We show that metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a well-documented AS-implicated lncRNA, regulates AS via two SFs, polypyrimidine tract-binding protein 1 (PTBP1) and PTB-associated SF (PSF). MALAT1 stabilizes the interaction between PTBP1 and PSF, thereby forming a functional module that affects a network of AS events. The MALAT1-stabilized PTBP1/PSF interaction occurs in multiple cellular contexts; however, the functional module, relative to MALAT1 only, has more dominant pathological significance in hepatocellular carcinoma. MALAT1 also stabilizes the PSF interaction with several heterogeneous nuclear ribonucleoparticle proteins other than PTBP1, hinting a broad role in AS control. We present a model in which MALAT1 cooperates with distinct SFs for AS regulation and pose that, relative to analyses exclusively performed for lncRNAs, a comprehensive consideration of lncRNAs and their binding partners may provide more information about their biological functions.
RESUMO
The early detection of biomarker proteins in clinical samples is of great significance for the diagnosis of diseases. However, it is still a challenge to detect low-concentration protein. Herein, a label-free aptamer-based amplification assay, termed the ATC-TA system, that allows fluorescence detection of very low numbers of protein without time-consuming washing steps and pre-treatment was developed. The target induces a conformational change in the allosteric aptasensor, triggers the target cycling and transcription amplification, and ultimately converts the input of the target protein into the output of the light-up aptamer (R-Pepper). It exhibits ultrahigh sensitivity with a detection limit of 5.62 fM at 37 â and the accuracy is comparable to conventional ELISA. ATC-TA has potential application for the detection of endogenous PDGF-BB in serum samples to distinguish tumor mice from healthy mice at an early stage. It also successfully detects exogenous SARS-CoV-2 spike proteins in human serum. Therefore, this high-sensitive, universality, easy-to-operate and cost-effective biosensing platform holds great clinical application potential in early clinical diagnosis.
RESUMO
MicroRNAs (miRNAs) are key players in human hepatocellular carcinoma (HCC) tumorigenesis. Therefore, small molecules targeting components of miRNA biogenesis may provide new therapeutic means for HCC treatment. By a high-throughput screening and structural simplification, we identified a small molecule, CIB-3b, which suppresses the growth and metastasis of HCC in vitro and in vivo by modulating expression profiles of miRNAome and proteome in HCC cells. Mechanistically, CIB-3b physically binds to transactivation response (TAR) RNA-binding protein 2 (TRBP) and disrupts the TRBP-Dicer interaction, thereby altering the activity of Dicer and mature miRNA production. Structure-activity relationship study via the synthesis of 45 CIB-3b derivatives showed that some compounds exhibited a similar inhibitory effect on miRNA biogenesis to CIB-3b. These results support TRBP as a potential therapeutic target in HCC and warrant further development of CIB-3b along with its analogues as a novel therapeutic strategy for the treatment of HCC.
Assuntos
Carcinoma Hepatocelular , RNA Helicases DEAD-box , Neoplasias Hepáticas , MicroRNAs , Coativadores de Receptor Nuclear , Ribonuclease III , Carcinoma Hepatocelular/tratamento farmacológico , Linhagem Celular , RNA Helicases DEAD-box/antagonistas & inibidores , Humanos , Neoplasias Hepáticas/tratamento farmacológico , MicroRNAs/metabolismo , Coativadores de Receptor Nuclear/antagonistas & inibidores , Proteínas de Ligação a RNA/metabolismo , Ribonuclease III/antagonistas & inibidoresRESUMO
Visualizing the dynamics of ATP in living cells is key to understanding cellular energy metabolism and related diseases. However, the live-cell applications of current methods are still limited due to challenges in biological compatibility and sensitivity to pH. Herein, a novel label-free fluorescent " turn-on " biosensor for monitoring ATP in living bacterias and mammalian cells was developed. This biosensor (Broc-ATP) employed heterobifunctional aptamers to detect ATP with high sensitivity in vitro. In our system, a very useful tandem method was established by combining four Broc-ATPs with 3 × F30 three-way junction scaffold to construct an intracellular biosensor that achieves sufficient fluorescence to respond to intracellular ATP. This intracellular biosensor can be used for sensitive and specific dynamic imaging of ATP in mammalian cells. Hence, this genetically encoded biosensor provides a robust and efficient tool for the detection of intracellular ATP dynamics and 3 × F30 tandem method expands the application of heterobifunctional aptamers in mammalian cells.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Trifosfato de Adenosina , Animais , Metabolismo Energético , Corantes Fluorescentes , OligonucleotídeosRESUMO
RNA-binding proteins (RBPs) are well-known to bind RNA via a set of RNA-binding domains (RBDs) and determine the fate and function of their RNA targets; inversely, some RBPs, in certain cases, may be modulated by the bound RNAs rather than regulate their RNA partners. Current proteome-wide studies reveal that almost half of RBPs have no canonical RBDs, and the discovery of tens of thousands of noncoding RNAs (ncRNAs), especially those with the size larger than 200 nt (namely long noncoding RNAs, lncRNAs), makes the crosstalk between RBPs and RNAs more complicated. It is clear that macromolecular complexes formed by RBP and RNA are not only a form of existence of their RBP and RNA components in cells, but also represent a functional entity through which those RBPs and regulatory ncRNAs participate in the construction of regulatory networks in organism. In this review, we summarize the multidimensional crosstalk between RBPs and ncRNAs in cancer and discuss how RBPs achieve their function via the bound ncRNAs in different aspects of gene expression as well as how RBPs direct modification and processing of ncRNAs, in order to better understand tumor biology and provide new insights into development of strategies for cancer therapy and early detection.
Assuntos
Biomarcadores Tumorais/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias/patologia , RNA Longo não Codificante/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Biomarcadores Tumorais/genética , Humanos , Neoplasias/genética , Neoplasias/metabolismo , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/genéticaRESUMO
A novel signal amplification to detect nucleic acid, called hairpin-mediated nicking enzymatic signal amplification (HNESA), is developed. This method overcomes the limitation of conventional nicking enzymatic signal amplification (NESA) that the target must contain the nicking endonuclease recognition site by using a hairpin probe containing the nicking endonuclease recognition site as an intermediary. Nucleic acid with any sequence can be amplified by HNESA which substantially improves the substrate-scope of traditional NESA. HNESA could detect nucleic acids (ssDNA and RNA) with a detection limit of 8.3 pM at 55 °C. As low as 68 fM could also be detected by integrating HNESA and strand-displacement amplification (SDA). More importantly, HNESA is quite efficient in distinguish single base mismatched sequences. HNESA has potential application for nucleic acid detection in complex biological samples. Therefore, HNESA with high sensitivity and ultrahigh selectivity, should be a promising tool for nucleic acid research, especially for single nucleotide polymorphism (SNP) detection.
Assuntos
Técnicas Biossensoriais , Ácidos Nucleicos , DNA de Cadeia Simples , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico , Ácidos Nucleicos/genética , RNARESUMO
Infections caused by drug-resistant "superbugs" pose an urgent public health threat due to the lack of effective drugs; however, certain mammalian proteins with intrinsic antibacterial activity might be underappreciated. Here, we reveal an antibacterial property against Gram-negative bacteria for factors VII, IX and X, three proteins with well-established roles in initiation of the coagulation cascade. These factors exert antibacterial function via their light chains (LCs). Unlike many antibacterial agents that target cell metabolism or the cytoplasmic membrane, the LCs act by hydrolyzing the major components of bacterial outer membrane, lipopolysaccharides, which are crucial for the survival of Gram-negative bacteria. The LC of factor VII exhibits in vitro efficacy towards all Gram-negative bacteria tested, including extensively drug-resistant (XDR) pathogens, at nanomolar concentrations. It is also highly effective in combating XDR Pseudomonas aeruginosa and Acinetobacter baumannii infections in vivo. Through decoding a unique mechanism whereby factors VII, IX and X behave as antimicrobial proteins, this study advances our understanding of the coagulation system in host defense, and suggests that these factors may participate in the pathogenesis of coagulation disorder-related diseases such as sepsis via their dual functions in blood coagulation and resistance to infection. Furthermore, this study may offer new strategies for combating Gram-negative "superbugs".
Assuntos
Farmacorresistência Bacteriana/efeitos dos fármacos , Fator IX/farmacologia , Fator VII/farmacologia , Fator X/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/fisiologia , Animais , Antibacterianos/farmacologia , Cromatografia Líquida de Alta Pressão , Fator IX/genética , Fator IX/metabolismo , Fator VII/genética , Fator VII/metabolismo , Fator X/genética , Fator X/metabolismo , Bactérias Gram-Negativas/fisiologia , Células Hep G2 , Humanos , Lipídeo A/análise , Lipídeo A/metabolismo , Lipopolissacarídeos/análise , Lipopolissacarídeos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/fisiologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Hammerhead ribozyme is the smallest and best characterized catalytic RNA-cleaving ribozyme. It has been reported as potential therapeutic tools to manipulate the expression of target genes. However, most of naturally occurring hammerhead ribozymes process self-cleavage rather than cleave substrate RNA in trans, and its high intracellular activity relies on the tertiary interaction of Loop II and steam I bulge, resulting in decreased performance as applied in gene silencing. We described a direct intracellular selection method to evolve hammerhead variants based on trans-cleavage mode via using a toxin gene as the reporter. And a dual fluorescence proteins system has also been established to quantitatively evaluate the efficiency of selected ribozymes in the cell. Based on this selection strategy, we obtained three mutants with enhanced intracellular cleaving activity compared to wide type hammerhead ribozyme. The best one, TX-2 was revealed to possess better and consistent gene knockdown ability at different positions on diverse targeted mRNA either in prokaryotic or eukaryotic cells than wild-type hammerhead ribozyme. These observations imply the efficiency of the intracellular selection method of the trans-acting ribozyme and the potentials of improved ribozyme variants for research and therapeutic purposes.
Assuntos
Conformação de Ácido Nucleico , RNA Catalítico/genética , RNA Mensageiro/genética , RNA/genética , Células Eucarióticas/metabolismo , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Inativação Gênica , Células Procarióticas/metabolismo , RNA/química , RNA Catalítico/química , RNA Catalítico/metabolismoRESUMO
Riboflavin (vitamin B2) has been thought to be a promising antitumoral agent in photodynamic therapy, though the further application of the method was limited by the unclear molecular mechanism. Our work reveals that riboflavin was able to recognize G-T mismatch specifically and induce single-strand breaks in duplex DNA targets efficiently under irradiation. In the presence of riboflavin, the photo-irradiation could induce the death of tumor cells that are defective in mismatch repair system selectively, highlighting the G-T mismatch as potential drug target for tumor cells. Moreover, riboflavin is a promising leading compound for further drug design due to its inherent specific recognition of the G-T mismatch.
Assuntos
Pareamento Incorreto de Bases/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Fotoquimioterapia/métodos , Riboflavina/uso terapêutico , Sequência de Bases , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/genética , Morte Celular/efeitos dos fármacos , Morte Celular/efeitos da radiação , Dano ao DNA/efeitos dos fármacos , Células HCT116 , Humanos , Luz , Neoplasias/patologia , Riboflavina/farmacologia , Riboflavina/efeitos da radiação , Especificidade por Substrato/efeitos dos fármacosRESUMO
A new guideline for the construction of hammerhead ribozymes to achieve trans-cleavage of a single-stranded RNA molecule was developed. The sequence rule of the HHRz cleavage site was highly recommended to be "DWH" with an optimal binding arm length of 8-9nt, which diverged from the former rule of "NUX".
Assuntos
Clivagem do RNA , RNA Catalítico/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , RNA Catalítico/genética , Schistosoma/enzimologiaRESUMO
Polymerase chain reaction coupled with signal generation offers sensitive recognition of target DNA sequence; however, these procedures require fluorophore-labeled oligonucleotide probes and high-tech equipment to achieve high specificity. Therefore, intensive research has been conducted to develop reliable, convenient, and economical DNA detection methods. The relay PCR described here is the first sequence-specific detection method using a small-molecule fluorophore as a sensor and combines the classic 5'-3' exonuclease activity of Taq polymerase with an RNA mimic of GFP to build a label-free DNA detection platform. Primarily, Taq polymerase cleaves the 5' noncomplementary overhang of the target specific probe during extension of the leading primer to release a relay oligo to initiate tandem PCR of the reporting template, which encodes the sequence of RNA aptamer. Afterward, the PCR product is transcribed to mRNA, which could generate a fluorescent signal in the presence of corresponding fluorophore. In addition to high sensitivity and specificity, the flexibility of choosing different fluorescent reporting signals makes this method versatile in either single or multiple target detection.
Assuntos
Técnicas Biossensoriais , DNA/metabolismo , Corantes Fluorescentes/química , Reação em Cadeia da Polimerase/métodosRESUMO
Constant efforts have been made to develop new method to realize sequence-specific RNA degradation, which could cause inhibition of the expression of targeted gene. Herein, by using an unmodified short DNA oligonucleotide for sequence recognition and endogenic small molecule, vitamin B2 (riboflavin) as photosensitizer, we report a simple strategy to realize the sequence-specific photocleavage of targeted RNA. The DNA strand is complimentary to the target sequence to form DNA/RNA duplex containing a G ⢠U wobble in the middle. The cleavage reaction goes through oxidative elimination mechanism at the nucleoside downstream of U of the G ⢠U wobble in duplex to obtain unnatural RNA terminal, and the whole process is under tight control by using light as switch, which means the cleavage could be carried out according to specific spatial and temporal requirements. The biocompatibility of this method makes the DNA strand in combination with riboflavin a promising molecular tool for RNA manipulation.
Assuntos
DNA/química , Fármacos Fotossensibilizantes/química , RNA/química , Riboflavina/química , Sequência de Bases/efeitos da radiação , Sítios de Ligação/efeitos da radiação , DNA/efeitos da radiação , Luz , Dados de Sequência Molecular , Fármacos Fotossensibilizantes/efeitos da radiação , RNA/efeitos da radiação , Riboflavina/efeitos da radiação , Relação Estrutura-AtividadeRESUMO
Single-nucleotide polymorphisms, either inherited or due to spontaneous DNA damage, are associated with numerous diseases. Developing tools for site-specific nucleotide modification may one day provide a way to alter disease polymorphisms. Here, we describe the in vitro selection and characterization of a new deoxyribozyme called F-8, which catalyzes nucleotide excision specifically at thymidine. Cleavage by F-8 generates 3'- and 5'-phosphate ends recognized by DNA modifying enzymes, which repair the targeted deoxyribonucleotide while maintaining the integrity of the rest of the sequence. These results illustrate the potential of DNAzymes as tools for DNA manipulation.
Assuntos
Clivagem do DNA , DNA Catalítico/química , DNA Catalítico/metabolismo , Timidina/metabolismo , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismoRESUMO
Ribozyme glmS based isothermal amplification assay is developed for the colorimetric detection of glucosamine-6-phosphate (GlcN6P). Upon binding to the metabolite target GlcN6P, self-cleavage of glmS ribozyme is initiated to release RNA fragment that can trigger the cascade signal amplification to release large amount of G-quadruplex DNAzymes as reporter for colorimetric detection. Given the importance of GlcN6P for cell wall biosynthesis, the glmS riboswitch has become a new drug target for the development of antibiotics. This assay not only offers a convenient detection of GlcN6P with high specificity and sensitivity, but also provides a platform for high-throughput screening of antibiotics based on glmS riboswitches.
Assuntos
Técnicas Biossensoriais/métodos , Glucosamina/análogos & derivados , Glucose-6-Fosfato/análogos & derivados , RNA Catalítico , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Colorimetria/métodos , Glucosamina/análise , Glucose-6-Fosfato/análise , Técnicas de Amplificação de Ácido Nucleico , RNA Catalítico/genéticaRESUMO
Fast and accurate detection of single-nucleotide polymorphism (SNP) is thought more and more important for understanding of human physiology and elucidating the molecular based diseases. A great deal of effort has been devoted to developing accurate, rapid, and cost-effective technologies for SNP analysis. However most of those methods developed to date incorporate complicated probe labeling and depend on advanced equipment. The DNAzyme based Gap-LCR detection method averts any chemical modification on probes and circumvents those problems by incorporating a short functional DNA sequence into one of LCR primers. Two kinds of exonuclease are utilized in our strategy to digest all the unreacted probes and release the DNAzymes embedded in the LCR product. The DNAzyme applied in our method is a versatile tool to report the result of SNP detection in colorimetric or fluorometric ways for different detection purposes.
Assuntos
Sequência de Bases/genética , DNA Catalítico/genética , Exonucleases/química , Polimorfismo de Nucleotídeo Único/genética , Colorimetria , DNA Catalítico/química , Genótipo , HumanosRESUMO
RNA detection has become an integral part of current biomedical research. Up to now, the reverse transcription-PCR has been the most practical method to detect mRNA targets. However, RNA detection by reverse transcription-PCR requires sophisticated equipment and it is highly sensitive to contamination with genomic DNA. Here we report a new isothermal reaction to simultaneously amplify and detect RNA, based on cleavage by DNAzyme and signal amplification. Cleavage-based signal amplification of RNA cannot be contaminated by genomic DNA and is suitable for the detection of both mRNA and microRNA targets, with high specificity and sensitivity. Moreover, the detection results can be reported in a colorimetric or real-time fluorometric way for different detection purposes.