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2.
Hum Gene Ther Methods ; 30(1): 1-16, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30700146

RESUMO

As a nucleic acid alternative to traditional antibody, aptamer holds great potential in various fields of biology and medicine such as targeted gene therapy, drug delivery, bio-sensing, and laboratory medicine. Over the past decades, the conventional Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method has undergone dramatic modifications and improvements owing to developments in material sciences and analytical techniques. However, many of the recently developed strategies either require complex materials and instruments or suffer from low efficiency and high failure rates in the selection of desired aptamers. Accordingly, the development of aptamers against new or novel targets is still a major obstacle for aptamer-based research and application. Here, an improved protein-SELEX procedure is presented for simplified and highly efficient isolation of aptamers against protein targets. Approaches are described that ensure a high success rate in aptamer selection by simplifying polymerase chain reaction procedures, introducing denature gel, utilizing an electro-elution-based single-stranded DNA separation strategy, as well as an enzyme-linked immunosorbent assay-based highly sensitive binding assay. In addition, a simplified sample preparation method for MiSeq-based next-generation sequencing is also introduced. While a recombinant protein as a bait protein for SELEX is discussed here, this protocol will also be invaluable for researchers wishing to develop aptamers against targets other than proteins such as small molecules, lipids, carbohydrates, cells, and micro-organisms for future gene therapy and/or diagnostics.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnica de Seleção de Aptâmeros/métodos , DNA de Cadeia Simples/isolamento & purificação , Ensaio de Imunoadsorção Enzimática/métodos , Biblioteca Gênica , Terapia Genética/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Reação em Cadeia da Polimerase , Proteínas Recombinantes
3.
Chem Commun (Camb) ; 54(36): 4593-4596, 2018 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-29670956

RESUMO

We have developed a novel functional nucleic acid aptamer to amyloid-ß peptide 1-40 (Aß1-40) and investigated its potential to detect Aß peptide fragments in neuropathologically confirmed Alzheimer brain hippocampus tissues samples. Our results demonstrate that the aptamer candidate RNV95 could detect tetrameric/pentameric low-molecular-weight Aß aggregates in autopsy hippocampal tissue from two neuropathologically confirmed Alzheimer disease cases. Although these are preliminary observations, detailed investigations are under way. This is the first demonstration of aptamer-Aß binding in Alzheimer brain tissues.


Assuntos
Doença de Alzheimer/diagnóstico por imagem , Peptídeos beta-Amiloides/metabolismo , Amiloide/metabolismo , Aptâmeros de Nucleotídeos/metabolismo , Hipocampo/patologia , Fragmentos de Peptídeos/metabolismo , Doença de Alzheimer/patologia , Amiloide/química , Peptídeos beta-Amiloides/química , Aptâmeros de Nucleotídeos/química , Humanos , Fragmentos de Peptídeos/química , Placa Amiloide/diagnóstico por imagem , Placa Amiloide/patologia , Agregação Patológica de Proteínas/metabolismo , Ligação Proteica , Multimerização Proteica
4.
Molecules ; 22(12)2017 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-29186905

RESUMO

Systematic evolution of ligands by exponential enrichment (SELEX) is an established procedure for developing short single-stranded nucleic acid ligands called aptamers against a target of choice. This approach has also been used for developing aptamers specific to whole cells named Cell-SELEX. Aptamers selected by Cell-SELEX have the potential to act as cell specific therapeutics, cell specific markers or cell specific drug delivery and imaging agents. However, aptamer development is a laborious and time-consuming process which is often challenging due to the requirement of frequent optimization of various steps involved in Cell-SELEX procedures. This review provides an insight into various procedures for selection, aptamer enrichment, regeneration and aptamer-binding analysis, in addition to a very recent update on all aptamers selected by Cell-SELEX procedures.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnica de Seleção de Aptâmeros/métodos , Bioensaio/métodos , Humanos , Ligação Proteica
5.
Sci Rep ; 7(1): 5898, 2017 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-28724889

RESUMO

The development of chemoresistance and inability in elimination of cancer stem cells are among the key limitations of cancer chemotherapy. Novel molecular therapeutic strategies able to overcome such limitations are urgently needed for future effective management of cancer. In this report, we show that EpCAM-aptamer-guided survivin RNAi effectively downregulated survivin both in colorectal cancer cells in vitro and in a mouse xenograft model for colorectal cancer. When combined with the conventional chemotherapeutic agents, the aptamer-guided survivin RNAi was able to enhance the sensitivity towards 5-FU or oxaliplatin in colorectal cancer stem cells, increase apoptosis, inhibit tumour growth and improve the overall survival of mice bearing xenograft colorectal cancer. Our results indicate that survivin is one of the key players responsible for the innate chemoresistance of colorectal cancer stem cells. Thus, aptamer-mediated targeting of survivin in cancer stem cells in combination with chemotherapeutic drugs constitutes a new avenue to improve treatment outcome in oncologic clinics.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Neoplasias Colorretais/patologia , Fluoruracila/farmacologia , Células-Tronco Neoplásicas/patologia , Interferência de RNA , Survivina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Sequência de Bases , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Regulação para Baixo/efeitos dos fármacos , Molécula de Adesão da Célula Epitelial/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Humanos , Camundongos SCID , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo
6.
Chembiochem ; 18(16): 1565-1567, 2017 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-28544018

RESUMO

Twice as apt: Nucleic acid aptamers with high binding affinity, specificity, epitope coverage and nuclease resistance were developed by using libraries containing oligonucleotides in which two bases in the pyrimidine nucleotide had been modified.


Assuntos
Aptâmeros de Nucleotídeos/química , Pró-Proteína Convertase 9/química , Nucleotídeos de Pirimidina/química , Animais , Aptâmeros de Nucleotídeos/síntese química , Desenho de Fármacos , Haplorrinos , Humanos , Lipoproteínas LDL/metabolismo , Camundongos , Inibidores de PCSK9 , Pró-Proteína Convertase 9/sangue , Nucleotídeos de Pirimidina/síntese química , Ratos
7.
Theranostics ; 5(12): 1456-72, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26681989

RESUMO

Understanding the molecular basis of drug resistance and utilising this information to overcome chemoresistance remains a key challenge in oncology. Here we report that survivin, a key protein implicated in drug resistance, is overexpressed in cancer stem cell pool of doxorubicin-resistant breast cancer cells. Moreover, by utilising an active targeting system consisting of an RNA aptamer targeted against the epithelial cell adhesion molecule and a Dicer substrate survivin siRNA, we could deliver a high dose of the siRNA to cancer stem cells in xenograft tumours. Importantly, silencing of survivin with this aptamer-siRNA chimera in cancer stem cell population led to the reversal of chemoresistance, such that combined treatment with low dose of doxorubicin inhibited stemness, eliminated cancer stem cells via apoptosis, suppressed tumour growth, and prolonged survival in mice bearing chemoresistant tumours. This strategy for in vivo cancer stem cell targeting has wide application for future effective silencing of anti-death genes and in fact any dysregulated genes involved in chemoresistance and tumour relapse.


Assuntos
Antígenos de Neoplasias/metabolismo , Antineoplásicos/farmacologia , Aptâmeros de Nucleotídeos/administração & dosagem , Moléculas de Adesão Celular/metabolismo , Doxorrubicina/farmacologia , Sinergismo Farmacológico , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Células-Tronco Neoplásicas/efeitos dos fármacos , Animais , Aptâmeros de Nucleotídeos/farmacocinética , Neoplasias da Mama/tratamento farmacológico , Modelos Animais de Doenças , Molécula de Adesão da Célula Epitelial , Feminino , Xenoenxertos/efeitos dos fármacos , Humanos , Camundongos SCID , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/farmacocinética , Survivina , Resultado do Tratamento
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