RESUMO
Benzo[a]pyrene-modified oligonucleotides were developed for the detection of RNAs with a point mutation. The probes produced two distinct fluorescence signals in response to single nucleotide differences in the RNA sequences, allowing for discrimination between the matched and single base mismatched RNA sequences in colorimetric and ratiometric manners.
Assuntos
Benzo(a)pireno , Corantes Fluorescentes , Mutação Puntual , RNA , Benzo(a)pireno/análise , Benzo(a)pireno/química , RNA/genética , RNA/química , RNA/análise , Corantes Fluorescentes/química , Colorimetria , Espectrometria de Fluorescência , Oligonucleotídeos/químicaRESUMO
A set of arylazopyrazole-based inhibitors targeting the mitotic motor protein CENP-E was discovered through the chemical platform using the quantitative cyclization of 1,3-diketone intermediate with various hydrazines under mild conditions. Through this efficient platform, the structure-activity relationship pertaining to the pyrazole photoswitch in photoswitchable CENP-E inhibitors not only in vitro but also in cells was successfully clarified.
Assuntos
Proteínas Cromossômicas não Histona , Pirazóis , Ciclização , Pirazóis/química , Pirazóis/farmacologia , Pirazóis/síntese química , Relação Estrutura-Atividade , Humanos , Proteínas Cromossômicas não Histona/antagonistas & inibidores , Proteínas Cromossômicas não Histona/metabolismo , Estrutura Molecular , Compostos Azo/química , Compostos Azo/farmacologia , Compostos Azo/síntese química , Relação Dose-Resposta a DrogaRESUMO
We developed a centromere-associated protein E (CENP-E) inhibitor employing trans to cis photoisomerization with 405 nm visible light illumination and fast thermal relaxation. This photoswitching characteristic of the inhibitor enabled selective blockage or release of the motion of particular chromosomes within a single mitotic cell. Using this technique, we successfully demonstrated targeted chromosome gain and loss in daughter cells by introducing asymmetric chromosome segregation.
Assuntos
Proteínas Cromossômicas não Histona , Luz , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/química , Humanos , Processos Fotoquímicos , Células HeLa , Compostos Azo/química , Compostos Azo/farmacologia , Estrutura Molecular , Segregação de Cromossomos/efeitos dos fármacosRESUMO
An arylazopyrazole-based covalent inhibitor targeting the mitotic motor protein of centromere-associated protein E (CENP-E) was developed. Using this photoswitchable inhibitor, a photoswitchable CENP-E was chemically constructed in cells, which enabled to local control of mitotic cell division with light illumination.
Assuntos
Proteínas Cromossômicas não Histona , Humanos , Proteínas Cromossômicas não Histona/antagonistas & inibidores , Proteínas Cromossômicas não Histona/metabolismo , Processos Fotoquímicos , Luz , Pirazóis/química , Pirazóis/farmacologia , Pirazóis/síntese química , Células HeLa , Estrutura Molecular , Mitose/efeitos dos fármacosRESUMO
The 332-nucleotide small nuclear RNA (snRNA) 7SK is a highly conserved non-coding RNA that regulates transcriptional elongation. By binding with positive transcriptional elongation factor b (P-TEFb) via HEXIM1, 7SK snRNA decreases the kinase activity of P-TEFb and inhibits transcriptional elongation. Additionally, it is reported that 7SK inhibition results in the stimulation of human immunodeficiency virus (HIV)-specific transcription. These reports suggest that 7SK is a naturally occurring functional molecule as negative regulator of P-TEFb and HIV transcription. In this study, we developed functional oligonucleotides that mimic the function of 7SK (7SK mimics) as novel inhibitors of HIV replication. We defined the essential region of 7SK regarding its suppressive effects on transcriptional downregulation using an antisense strategy. Based on the results, we designed 7SK mimics containing the defined region. The inhibitory effects of 7SK mimics on HIV-1 long terminal repeat promoter specific transcription was drastic compared with those of the control mimic molecule. Notably, these effects were found to be more enhanced by co-transfection with Tat-expressing plasmids. From these results, it is indicated that 7SK mimics may have great therapeutic potential for HIV/AIDS treatment.
Assuntos
Desenvolvimento de Medicamentos , RNA Nuclear Pequeno/farmacologia , Transcrição Gênica/efeitos dos fármacos , Produtos do Gene tat do Vírus da Imunodeficiência Humana/antagonistas & inibidores , Relação Dose-Resposta a Droga , Estrutura Molecular , RNA Nuclear Pequeno/síntese química , RNA Nuclear Pequeno/química , Relação Estrutura-Atividade , Transcrição Gênica/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genéticaRESUMO
Nucleic acids in body fluids, such as circulating cell-free nucleic acids, viral DNA, and RNA have received much attention for their great potential as biomarkers in liquid biopsies of serious diseases. Although quantitative polymerase chain reaction (qPCR) has been traditionally used as a laboratory-based assay for measuring nucleic acids, there is a strong demand for techniques to qualitatively, rapidly, and simply measure the extremely low-abundance nucleic acids in order to realize the nucleic acid-based liquid biopsies. With this aim in mind, we developed a simple and highly sensitive sandwich-type assay for nucleic acids using a combination of surface-enhanced Raman scattering (SERS), which enhances Raman scattering by 108- to 1010-fold, and bioorthogonal Raman tags, which generate signals in the biologically silent region (1800-2800 cm-1). Using gold nanorods having approximately 240 strands of oligonucleotides and 4-cyano-N-(2-mercaptoethyl)benzamide (4CMB) as the bioorthogonal Raman tag, we successfully detected target nucleic acids in a sequence-selective manner.
Assuntos
DNA/análise , Análise Espectral Raman/métodos , DNA/química , Ouro/química , Microesferas , Nanotubos/química , Hibridização de Ácido Nucleico , Sefarose/química , Propriedades de SuperfícieRESUMO
Self-assembled peptide nanofibers (NFs) obtained from ß-sheet peptides conjugated with drugs, including antigenic peptides, have recently attracted significant attention. However, extensive studies on the interactions of ß-sheet peptide NFs with model cell membranes have not been reported. In this study, we investigated the interactions between three types of NFs, composed of PEG-peptide conjugates with different ethylene glycol (EG) lengths (6-, 12- and 24-mer), and dipalmitoylphosphatidylcholine (DPPC) Langmuir membranes. When increasing the EG chain length, those interactions significantly decreased considering measurements in the presence of the NFs of: (i) changes in surface pressure of the DPPC Langmuir monolayers and (ii) surface pressure-area (π-A) compression isotherms of DPPC. Because the observed trend was similar to the EG length dependency with regard to cellular association and cytotoxicity of the NFs that was reported previously, the interaction of NFs with phospholipid membranes represented a crucial factor to determine the cellular association and toxicity of the NFs. In contrast to NFs, no changes were observed with varying EG chain length on the interaction of the building block peptide with the DPPC membrane. The results obtained herein can provide a design guideline on the formulation of ß-sheet peptide NFs, which may broaden its potential.
Assuntos
Membranas Artificiais , Nanofibras/química , Etilenoglicol/químicaRESUMO
MicroRNAs in exosomes (exosomal miRNAs) are considered as significant targets for cancer therapy. Anti-miR oligonucleotides are often used for the functional inhibition of miRNAs; however, there are no studies regarding the regulation of exosomal miRNA functions. In this study, we attempted to develop a novel drug delivery system using anti-exosome antibody-anti-miR oligonucleotide complexes (ExomiR-Tracker) to hijack exosomes to carry anti-miR oligonucleotides inside exosome-recipient cells. We found that ExomiR-Tracker bound to the exosomes, and then the complexes were introduced into the recipient cells. We also found that anti-miR oligonucleotides introduced into the recipient cells can exhibit inhibitory effects on exosomal miRNA functions in vitro and in vivo. We believe that our strategy would be a promising one for targeting exosomal miRNAs.
RESUMO
Point mutations are well characterized activators of oncogenes but are often indistinguishable using common gene technologies. In general, the precise sites of point-mutated oncogenes are difficult to distinguish under physiological conditions primarily because single nucleotide mismatch do not affect the annealing temperatures of DNA probes sufficiently. To address this limitation, we developed photo-responsive oligodeoxyribonucleotides containing 2'-O-[N-(4,5',8-trimethylpsoralen-4'-ylmethylcarbamoyl)]adenosine (Ps-amd-Oligo), which can be used to selectively manipulate and identify genes with point mutations. Here we present time course analyses of the photo-cross-linking efficiency of Ps-amd-Oligo with DNA and RNA and show promising selectivity for the oncogene H-ras.
Assuntos
Adenosina/análogos & derivados , Adenosina/química , DNA , Mutação , Oligodesoxirribonucleotídeos/química , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/genética , Adenosina/síntese química , Reagentes de Ligações Cruzadas , DNA/química , DNA/genética , Estrutura Molecular , Oligodesoxirribonucleotídeos/genética , Mutação Puntual , Raios UltravioletaRESUMO
Recently, nanofibers (NFs) formed from antigenic peptides conjugated to ß-sheet-forming peptides have attracted much attention as a new generation of vaccines. However, studies describing how the hydrophilic-hydrophobic balance of NF components affects cellular interactions of NFs are limited. In this report, three different NFs were prepared by self-assembly of ß-sheet-forming peptides conjugated with model antigenic peptides (SIINFEKL) from ovalbumin and hydrophilic oligo-ethylene glycol (EG) of differing chain lengths (6-, 12- and 24-mer) to investigate the effect of EG length of antigen-loaded NFs on their cellular uptake, cytotoxicity, and dendritic cell (DC)-stimulation ability. We used an immortal DC line, termed JAWS II, derived from bone marrow-derived DCs of a C57BL/6 p53-knockout mouse. The uptake of NFs, consisting of the EG 12-mer by DCs, was the most effective and activated DC without exhibiting significant cytotoxicity. Increasing the EG chain length significantly reduced cellular entry and DC activation by NFs. Conversely, shortening the EG chain enhanced DC activation but increased toxicity and impaired water-dispersibility, resulting in low cellular uptake. These results show that the interaction of antigen-loaded NFs with cells can be tuned by the EG length, which provides useful design guidelines for the development of effective NF-based vaccines.
Assuntos
Adjuvantes Imunológicos/farmacologia , Antígenos/farmacologia , Células Dendríticas/efeitos dos fármacos , Ovalbumina/farmacologia , Peptídeos/farmacologia , Adjuvantes Imunológicos/química , Sequência de Aminoácidos , Animais , Antígenos/química , Linhagem Celular , Células Cultivadas , Células Dendríticas/imunologia , Etilenoglicol/química , Etilenoglicol/farmacologia , Interações Hidrofóbicas e Hidrofílicas , Camundongos Endogâmicos C57BL , Nanofibras/química , Nanofibras/ultraestrutura , Ovalbumina/química , Peptídeos/química , Conformação Proteica em Folha betaRESUMO
MicroRNAs (miRNAs) regulate gene expression by forming RNA-induced silencing complexes (RISCs) and have been considered as promising therapeutic targets. MiRNA is an essential component of RISC for the modulation of gene expression. Therefore, the release of miRNA from RISC is considered as an effective method for the inhibition of miRNA functions. In our previous study, we reported that anti-miRNA oligonucleotides (AMOs), which are composed of the 2'-O-methyl (2'-OMe) RNA, could induce the release of miRNA from RISC. However, the mechanisms underlying the miRNA-releasing effects of chemically modified AMOs, which are conventionally used as anti-cancer drugs, are still unclear. In this study, we investigated the relationship between the miRNA releasing rate from RISC and the inhibitory effect on RISC activity (IC50) using conventional chemically modified AMOs. We demonstrated that the miRNA-releasing effects of AMOs are directly proportional to the IC50 values, and AMOs, which have an ability to promote the release of miRNA from RISC, can effectively inhibit RISC activity in living cells.
Assuntos
Antagomirs/metabolismo , MicroRNAs/metabolismo , Complexo de Inativação Induzido por RNA/antagonistas & inibidores , Complexo de Inativação Induzido por RNA/metabolismo , Antagomirs/genética , Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Concentração Inibidora 50 , MicroRNAs/genéticaRESUMO
Because point mutations in GTPase-coding genes have been reported to be responsible for the transformation of cells, anticancer reagents that react effectively and sequence selectively with target RNAs having a point mutation are highly desired. In this study, we developed novel photo-cross-linking oligodeoxyribonucleotides ((pro)PCA-ODNs) that had a caged α-chloroaldehyde group conjugated to a 2-methylpropanediyl backbone ((pro)PCA) in the middle of the strand. A kinetic study of the deprotection reaction of (pro)PCA-ODN revealed that the bis(2-nitrobenzyl)acetal group was completely deprotected within 1 min. Photo-cross-linking studies of (pro)PCA-ODNs with complementary oligoribonucleotides (ORNs) revealed that (pro)PCA-ODNs reacts efficiently and selectively with the target ORNs that have an adenosine or cytidine residue at a frontal position of the (pro)PCA residue without adverse effects of bases adjacent to the mutation site.
Assuntos
Adenosina/química , Aldeídos/química , GTP Fosfo-Hidrolases/química , Indicadores e Reagentes/química , Oligodesoxirribonucleotídeos Antissenso/química , Mutação Puntual/genética , RNA/química , GTP Fosfo-Hidrolases/genética , Cinética , RNA/genéticaRESUMO
MicroRNAs (miRNAs) are known to be important post-transcription regulators of gene expression. Aberrant miRNA expression is associated with pathological disease processes, including carcinogenesis. Therefore, miRNAs are considered significant therapeutic targets for cancer therapy. MiRNAs do not act alone, but exhibit their functions by forming RNA-induced silencing complex (RISC). Thus, the regulation of RISC activity is a promising approach for cancer therapy. MiRNA is a core component of RISC and is an essential to RISC for recognizing target mRNA. Thereby, it is expected that development of the method to promote the release of miRNA from RISC would be an effective approach for inhibition of RISC activity. In this study, we synthesized novel peptide-conjugated oligonucleotides (RINDA-as) to promote the release of miRNA from RISC. RINDA-as showed a high rate of miRNA release from RISC and high level of inhibitory effect on RISC activity.
Assuntos
MicroRNAs/metabolismo , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/farmacologia , Peptídeos/química , Peptídeos/farmacologia , Complexo de Inativação Induzido por RNA/antagonistas & inibidores , Complexo de Inativação Induzido por RNA/metabolismo , Sequência de Bases , Células HEK293 , Células HeLa , HumanosRESUMO
4,5',8-Trimethylpsoralen-conjugated oligonucleotides have been used in the study of photo-cross-linking with target oligonucleotides and in the field of the photodynamic therapy. This unit describes synthetic procedures for oligonucleotides using 2'-O-methylphosphoramidite units and an adenosine phosphoramidite unit containing a 4,5',8-trimethylpsoralen derivative attached at the 2' position of an adenosine sugar moiety via an ethoxymethylene linkage. Procedures for obtaining the photo-cross-linking efficiency of 2'-O-methyloligonucleotides containing a 4,5',8-trimethylpsoralen derivative with a target oligonucleotide under UV irradiation conditions are also described, together with the procedure for preparation of (32)P-radiolabeled RNA.
Assuntos
Reagentes de Ligações Cruzadas/química , Oligonucleotídeos/química , Oligonucleotídeos/síntese química , RNA/química , Trioxsaleno/química , Raios UltravioletaRESUMO
We developed a novel method for regulation of RISC function by photoreactive oligonucleotides (Ps-Oligo) containing 2'-O-psoralenylmethoxyethyl adenosine (Aps). We observed that inhibitory effects of Ps-Oligos on RISC function were enhanced by UV-irradiation compared with 2'-O-methyl-oligonucleotide without Aps. These results suggest Ps-Oligo inhibited RISC function by cross-linking effect, and we propose that the concept described in this report may be promising and applicable one to regulate the small RNA-mediated post-transcriptional regulation.
Assuntos
Ficusina/química , Oligonucleotídeos/química , Oligonucleotídeos/farmacocinética , Complexo de Inativação Induzido por RNA/genética , Adenosina , Reagentes de Ligações Cruzadas/química , Regulação da Expressão Gênica/efeitos dos fármacos , Células HeLa/efeitos dos fármacos , Células HeLa/efeitos da radiação , Humanos , Oligonucleotídeos Antissenso/farmacologia , Fotoquímica/métodos , Complexo de Inativação Induzido por RNA/química , Termodinâmica , Thermus thermophilus/genética , Raios UltravioletaRESUMO
DNA methylation and demethylation significantly affect the deactivation and activation processes of gene expression significantly. In particular, C-5-methylation of cytosine in the CpG islands is important for the epigenetic modification in genes, which plays a key role in regulating gene expression. The determination of the location and frequency of DNA methylation is important for the elucidation of the mechanisms of cell differentiation and carcinogenesis. Here we designed a psoralen-conjugated oligonucleotide (PS-oligo) for the discrimination of 5-methylcytosine (5-mC) in DNA. The cross-linking behavior of psoralen derivatives with pyrimidine bases, such as thymine, uracil and cytosine has been well discussed, but there are no reports which have examined whether cross-linking efficiency of psoralen with cytosine would be changed with or without C-5 methylation. We found that the cross-linking efficiency of PS-oligo with target-DNA containing 5-mC was greatly increased compared to the case of target-DNA without 5-mC, approximately seven-fold higher. Here we report a new aspect of the photocross-linking behavior of psoralen with 5-mC that is applicable to a simple, sequence-specific and quantitative analysis for the discrimination of 5-mC in DNA, which can be applicable to study the epigenetic behavior of gene expressions.
Assuntos
Citosina/química , Metilação de DNA , Furocumarinas/química , Oligonucleotídeos/química , FotoquimioterapiaRESUMO
Hereditary coproporphyria (HCP) is an autosomal dominant-inherited disease of haem biosynthesis caused by partial deficiency of the enzyme coproporphyrinogen oxidase (CPOX). Patients with HCP show <50% of normal activity and those with the rare autosomal recessive harderoporphyria accumulate harderoporphyrinogen, an intermediate porphyrin of the CPOX reaction. To clarify the relationship of the low enzyme activity with these diseases, we expressed mutant CPOX carrying His-tag from these porphyria patients and co-expressed mutant CPOX carrying His-tag and normal CPOX carrying HA-tag in a tandem fashion in Escherichia coli. Purification of the His-tag-containing enzyme revealed that the His-enzyme forms a heterodimer in association with the HA-enzyme, and analysis using a cross-link reagent confirmed that the enzyme is a dimer (â¼70 kDa). Then, we expressed homo- and heterodimers composed of the wild-type (wt) and engineered mutants of the enzyme or mutants from HCP patients. The monomer form of mutated CPOX did not show any activity and homodimeric enzymes derived from HCP mutant showed low activity (<20% of the control). Some mutations of amino acids 401-404 were associated with marked accumulation of harderoporphyrinogen, with a decrease in the production of protoporphyrinogen, whereas K404E derived from patients with harderoporphyria produced less harderoporphyrinogen. The heterodimers with wt and mutated subunits from HCP patients showed low protoporphyrinogen producing activity. These results show that the substitution of amino acids from R401 to K404 results in extremely low enzyme activity with either mutant homodimer or heterodimers containing normal and mutated subunits and can be linked to HCP disease.
Assuntos
Coproporfiria Hereditária/enzimologia , Coproporfirinogênio Oxidase/química , Coproporfirinogênio Oxidase/genética , Mutação , Engenharia de Proteínas , Multimerização Proteica/genética , Coproporfiria Hereditária/genética , Coproporfirinogênio Oxidase/metabolismo , HumanosRESUMO
We developed a photoresponsive α-bromoaldehyde-conjugated oligonucleotide (PBA-ODN). The PBA-ODN selectively reacted and formed covalent bonds with target oligonucleotides having adenine or cytosine at the frontal position of the aldehyde derivative. Kinetic studies revealed that PBA-ODN has increased kinetic rates for the formation of cross-linked duplexes compared with the corresponding α-chloroaldehyde-conjugated oligonucleotide (PCA-ODN).
Assuntos
Aldeídos/química , Reagentes de Ligações Cruzadas/química , Oligonucleotídeos/química , Halogenação , CinéticaRESUMO
Fluorescent probes are powerful and indispensable tools for imaging RNA in vivo and in vitro. To simultaneously visualize multiple RNA targets in a cell, it is necessary to develop probes which emit fluorescence with different colors by excitation at a single wavelength. We synthesized OMUpy1 and OMUpy2 in this study with a cyanine dye respectively conjugated at their 5' ends. A fluorescent analysis revealed these probes to have yellow or pink fluorescence derived from the cyanine dyes with an extremely large Stokes shift. Three color-coded fluorescent images were also obtained in the presence of target RNAs.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , RNA/química , Carbocianinas/química , Oligorribonucleotídeos/químicaRESUMO
Recent RNA research has revealed the close involvement of various RNAs in cellular functions. RNAs are becoming the inevitable target molecules for research into details of gene expression. RNA and its related complexes are also promising targets for disease diagnosis. Multi cellular specimens such as organ tissues, histopathological specimens, and embryos are among the possible targets of RNA-based diagnostic techniques. In this report, we focused on a method that would provide such spatial and temporal information. We demonstrated that an RNA-specific probe (OMUpy2) was not only applicable to the detection of a specific mRNA in Drosophila embryos in a temporal and spatial manner but was also relatively quick and easy to use. The probe, OMUpy2, could be applied to other multi cellular systems for RNA-based diagnosis and research. The promising results of this manuscript show the great potential of RNA-based detection for both biological research and diagnostic medicine.