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1.
Nucleic Acids Res ; 51(16): 8880-8890, 2023 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-37503836

RESUMO

Ligand/protein molecular recognition involves a dynamic process, whereby both partners require a degree of structural plasticity to regulate the binding/unbinding event. Here, we present the characterization of the interaction between a highly dynamic G-rich oligonucleotide, M08s-1, and its target protein, human α-thrombin. M08s-1 is the most active anticoagulant aptamer selected thus far. Circular dichroism and gel electrophoresis analyses indicate that both intramolecular and intermolecular G-quadruplex structures are populated in solution. The presence of thrombin stabilises the antiparallel intramolecular chair-like G-quadruplex conformation, that provides by far the main contribution to the biological activity of the aptamer. The crystal structure of the thrombin-oligonucleotide complex reveals that M08s-1 adopts a kinked structural organization formed by a G-quadruplex domain and a long duplex module, linked by a stretch of five purine bases. The quadruplex motif hooks the exosite I region of thrombin and the duplex region is folded towards the surface of the protein. This structural feature, which has never been observed in other anti-exosite I aptamers with a shorter duplex motif, hinders the approach of a protein substrate to the active site region and may well explain the significant increase in the anticoagulant activity of M08s-1 compared to the other anti-exosite I aptamers.


Assuntos
Anticoagulantes , Aptâmeros de Nucleotídeos , Trombina , Humanos , Anticoagulantes/química , Aptâmeros de Nucleotídeos/química , Dicroísmo Circular , Quadruplex G , Guanina/química , Trombina/química
2.
Anal Biochem ; 687: 115452, 2024 04.
Artigo em Inglês | MEDLINE | ID: mdl-38158105

RESUMO

Low-molecular drug discovery using DNA-encoded chemical library (DEL) is a powerful technology, although improving the partitioning efficiency of affinity ligands from DEL remains a challenge. Here, we assessed the usefulness of microbead-assisted capillary electrophoresis (MACE) for partitioning peptide-oligonucleotide conjugates (POCs), in which high selection pressure is applied because of different mobility of target-modified beads and POCs during CE. Despite their different charge characteristics, all POCs were well separated from the beads. When bead extraction was performed, the tagged DNA amplification was observed only in the couple of a ligand/target, suggesting proficiently specific partitioning of peptide ligands was accomplished using MACE.


Assuntos
Oligonucleotídeos , Peptídeos , Microesferas , Peptídeos/química , Oligonucleotídeos/química , Eletroforese Capilar/métodos , DNA/química , DNA de Cadeia Simples
3.
Cell Tissue Res ; 384(3): 643-653, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33783611

RESUMO

Individual olfactory sensory neurons (OSNs) in the mouse main olfactory epithelium express a single odorant receptor (OR) gene from the repertoire of either class I or class II ORs. The transcription factor Bcl11b determines the OR class to be expressed in OSNs. The septal organ (SO), a small neuroepithelium located at the ventral base of the nasal septum, is considered as an olfactory subsystem because it expresses a specific subset of ORs. However, the mechanisms underlying the generation and differentiation of SO-OSN remain unknown. In the present study, we show that the generation and differentiation of SO-OSN employ the same genetic pathway as in the OSN lineage, which is initiated by the neuronal fate determinant factor Ascl1. Additionally, the key role of Bcl11b in the SO is demonstrated by the abnormal phenotypes of Bcl11b-deficient mice: significant reduction in the expression of OR genes and in the number of mature SO-OSNs. Although SO-OSNs are specified to express a subset of class II OR genes in wild-type mice, the Bcl11b deletion led to the expression of class I OR genes, while the expression of class II OR genes was significantly decreased, with one exception of Olfr15. These results indicate that Bcl11b is necessary for proper OR expression in SO-OSNs.


Assuntos
Perfuração do Septo Nasal/metabolismo , Mucosa Olfatória/metabolismo , Neurônios Receptores Olfatórios/metabolismo , Receptores Odorantes/metabolismo , Proteínas Repressoras/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucosa Olfatória/citologia , Neurônios Receptores Olfatórios/citologia , Olfato
4.
Analyst ; 142(21): 4030-4038, 2017 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-28875191

RESUMO

A single-round DNA aptamer selection for mammalian cells was successfully achieved for the first time using a capillary electrophoresis (CE)-based methodology called polymer-enhanced capillary transient isotachophoresis (PectI). The PectI separation yielded a single peak for the human lung cancer cell line (PC-9) complexed with DNA aptamer candidates, which was effectively separated from a free randomized DNA library peak, ensuring no contamination from free DNA in the PC-9-DNA aptamer complex fraction. The DNA aptamer candidates obtained after a single-round selection employing counter selection with HL-60 were proven to bind selectively and form kinetically stable complexes with PC-9 cells. Interestingly, most aptamer candidates showed high binding ability (Kd = 70-350 nM) with different extents of binding on the cell surface. These facts proved that a single-round selection for mammalian cells by PectI is feasible to obtain various types of aptamer candidates, which have high-affinity even for non-overexpressed but unique targets on the cell surface in addition to overexpressed targets.


Assuntos
Aptâmeros de Nucleotídeos , Eletroforese Capilar , Biblioteca Gênica , Isotacoforese , Linhagem Celular Tumoral , Humanos , Polímeros
5.
Mol Ther Nucleic Acids ; 33: 762-772, 2023 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-37621412

RESUMO

Heparin-induced thrombocytopenia (HIT) is a complication caused by administration of the anticoagulant heparin. Although the number of patients with HIT has drastically increased because of coronavirus disease 2019 (COVID-19), the currently used thrombin inhibitors for HIT therapy do not have antidotes to arrest the severe bleeding that occurs as a side effect; therefore, establishment of safer treatments for HIT patients is imperative. Here, we devised a potent thrombin inhibitor based on bivalent aptamers with a higher safety profile via combination with the antidote. Using an anti-thrombin DNA aptamer M08s-1 as a promising anticoagulant, its homodimer and heterodimer with TBA29 linked by a conformationally flexible linker or a rigid duplex linker were designed. The dimerized M08s-1-based aptamers had about 100-fold increased binding affinity to human and mouse thrombin compared with the monomer counterparts. Administration of these bivalent aptamers into mice revealed that the anticoagulant activity of the dimers significantly surpassed that of an approved drug for HIT treatment, argatroban. Moreover, adding protamine sulfate as an antidote against the most potent bivalent aptamer completely suppressed the anticoagulant activity of the dimer. Emerging potent and neutralizable anticoagulant aptamers will be promising candidates for HIT treatment with a higher safety profile.

6.
Res Pract Thromb Haemost ; 5(5): e12503, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34136744

RESUMO

BACKGROUND: Bivalent thrombin-binding aptamers (TBAs) have great potential for the treatment of thrombosis because they exhibit high anticoagulant activity, and their complementary single-stranded DNA (ssDNA) sequences work as an antidote. However, a design strategy for antidote sequences against bivalent aptamers has not been established. OBJECTIVES: To develop bivalent TBAs using M08, which exhibits higher anticoagulant activity than the previously reported exosite Ⅰ-binding DNA aptamers, such as HD1, an exosite Ⅱ-binding DNA aptamer (HD22) was linked to M08 with various types of linkers. In addition, short-length complementary ssDNAs were designed to neutralize the optimized bivalent aptamer effectively and rapidly. RESULTS: Among the bivalent aptamers of M08 linked to HD22 with various types of linkers, M08-T15-HD22 possessed approximately 5-fold higher anticoagulant activity than previously reported bivalent aptamers. To neutralize the activity of the 87-meric M08-T15-HD22, complementary ssDNA sequences with different lengths and hybridization segments were designed. The complementary sequence against the M08 moiety played a more important role in neutralizing than that against the HD22 moiety. Hybridization of the T15 linker in the M08-T15-HD22 with the A15 sequence in the antidote accelerated neutralization due to toehold-mediated strand displacement. Interestingly, some shorter-length antidotes showed higher neutralizing activity than the full complementary 87-meric antidote, and the shortest, 34-meric antidote, neutralized most effectively. CONCLUSIONS: A pair comprising an 87-meric bivalent TBA containing M08 and a 34-meric short-length antidote with high anticoagulant and rapid neutralizing activities was developed. This design strategy of the DNA sequence can be used for other bivalent DNA aptamers and their antidotes.

7.
Anal Sci ; 35(5): 585-588, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31080213

RESUMO

Nucleobase-modified aptamers are attractive candidates for diagnostic and therapeutic agents due to the high affinity, stability and functionality. However, since even conventional SELEX requires many selection rounds, acquisition of modified aptamers is much more laborious. Herein, microbeads-assisted capillary electrophoresis (MACE)-SELEX was applied against thrombin using the indole-modified DNA library. After only three selection rounds, we successfully enriched the modified aptamers and they showed slower off-rate than reported aptamers, suggesting MACE-SELEX is a promising approach for rapid identification of modified aptamers.


Assuntos
Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/isolamento & purificação , DNA/química , Eletroforese Capilar/métodos , Microesferas , Técnica de Seleção de Aptâmeros , Aptâmeros de Nucleotídeos/genética , DNA/genética , Biblioteca Gênica , Humanos , Indóis/farmacologia , Trombina/antagonistas & inibidores
8.
Anal Sci ; 35(1): 113-116, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30626772

RESUMO

Here, we demonstrated a strategy for developing signaling aptamers, based on screening of signaling aptamers from multiple aptamer candidates obtained by SELEX with next generation sequencing. Among aptamer candidates labelled by 6-carboxyfluorescein and quencher at both end termini, there is the possibility of discovering a potent signaling aptamer. In this study, we discovered DNA signaling aptamers against VEGFR-1. This strategy has the potential for signaling aptamer discovery without the extremely laborious task of optimization of oligodeoxynucleotide modifications.


Assuntos
Aptâmeros de Nucleotídeos/química , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Técnica de Seleção de Aptâmeros/métodos , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/metabolismo , Sequência de Bases , Biblioteca Gênica , Ligação Proteica , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/análise , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/análise , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
9.
Mol Ther Nucleic Acids ; 16: 348-359, 2019 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-30986696

RESUMO

We present a rapidly neutralizable and highly anticoagulant thrombin-binding aptamer with a short toehold sequence, originally discovered by systematic evolution of ligands by exponential enrichment (SELEX) with microbead-assisted capillary electrophoresis (MACE). MACE is a novel CE-partitioning method for SELEX and able to separate aptamers from a library of unbound nucleic acids, where the aptamer and target complexes can be detected reliably and partitioned with high purity even in the first selection cycle. Three selection rounds of MACE-SELEX discovered several TBAs with a nanomolar affinity (Kd = 4.5-8.2 nM) that surpasses previously reported TBAs such as HD1, HD22, and NU172 (Kd = 118, 13, and 12 nM, respectively). One of the obtained aptamers, M08, showed a 10- to 20-fold longer prolonged clotting time than other anticoagulant TBAs, such as HD1, NU172, RE31, and RA36. Analyses of the aptamer and thrombin complexes using both bare and coated capillaries suggested that a large number of efficient aptamers are missed in conventional CE-SELEX because of increased interaction between the complex and the capillary. In addition, the toehold-mediated rapid antidote was designed for safe administration. The efficient aptamer and antidote system developed in the present study could serve as a new candidate for anticoagulant therapy.

10.
Chem Commun (Camb) ; 52(3): 461-4, 2016 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-26525483

RESUMO

We present a polymer-enhanced capillary transient isotachophoresis (PectI) selection methodology for acquisition of high-affinity (kinetically inert) DNA aptamers capable of recognizing distinct microbial cell surfaces, which requires only a single electrophoretic separation between particles (free cells and cells bound with aptamers) and molecules (unbound or dissociated DNA) in free solution.


Assuntos
Aptâmeros de Nucleotídeos/química , Bactérias/química , Motivos de Nucleotídeos , Sequência de Bases , Bioensaio , Isotacoforese , Microscopia de Fluorescência , Dados de Sequência Molecular , Fatores de Tempo
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