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1.
Talanta ; 193: 110-117, 2019 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30368278

RESUMO

We report a novel method for efficiently screening aptamers from a complex ssDNA library based on silver decahedral nanoparticles (AgNP) and fluorescence activated cell sorting (FACS). In this method, target protein (lactoferrin) and negative proteins (α-lactalbumin, ß-lactoglobulin, bovine serum albumin, casein) were respectively immobilized on polystyrene microspheres (PS) to form PSLac, PSα-Lac, PSß-Lac, PSBSA and PSCas. PSLac was firstly interacted with Cy5 labeled library (Lib), then hybridized with Cy5 modified silver decahedral nanoparticles (AgNPCy5) to form PSLac/Lib/AgNPCy5 conjugates. FACS was used to separate and collect PSLac/Lib/AgNPCy5 conjugates from complicated complex. AgNP was used to increase the fluorescence intensity in the selecting process and choose non-self-hybridization of Lib. Six aptamers (Ylac1, Ylac4, Ylac5, Ylac6, Ylac8 and Ylac9) were obtained after five-round of selection. These aptamers showed good specificity towards lactoferrin in the presence of negative proteins. The equilibrium dissociation constants (Kd) of six aptamers were calculated and all were in the nanomolar range. In a word, AgNP-FACS SELEX (AgFACS-SELEX) is a rapid, sensitive and highly efficient method for screening aptamers.


Assuntos
Aptâmeros de Nucleotídeos/química , Citometria de Fluxo/métodos , Lactoferrina/química , Nanopartículas Metálicas/química , Técnica de Seleção de Aptâmeros/métodos , Prata/química , Adsorção , Animais , Sequência de Bases , Carbocianinas/química , Bovinos , Linhagem Celular , DNA de Cadeia Simples/química , Fluorescência , Corantes Fluorescentes/química , Humanos , Poliestirenos/química
2.
Nat Med ; 18(2): 307-14, 2012 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-22286306

RESUMO

Metabolic skeletal disorders associated with impaired bone formation are a major clinical challenge. One approach to treat these defects is to silence bone-formation-inhibitory genes by small interference RNAs (siRNAs) in osteogenic-lineage cells that occupy the niche surrounding the bone-formation surfaces. We developed a targeting system involving dioleoyl trimethylammonium propane (DOTAP)-based cationic liposomes attached to six repetitive sequences of aspartate, serine, serine ((AspSerSer)(6)) for delivering siRNAs specifically to bone-formation surfaces. Using this system, we encapsulated an osteogenic siRNA that targets casein kinase-2 interacting protein-1 (encoded by Plekho1, also known as Plekho1). In vivo systemic delivery of Plekho1 siRNA in rats using our system resulted in the selective enrichment of the siRNAs in osteogenic cells and the subsequent depletion of Plekho1. A bioimaging analysis further showed that this approach markedly promoted bone formation, enhanced the bone micro-architecture and increased the bone mass in both healthy and osteoporotic rats. These results indicate (AspSerSer)(6)-liposome as a promising targeted delivery system for RNA interference-based bone anabolic therapy.


Assuntos
Anabolizantes/uso terapêutico , Sistemas de Liberação de Medicamentos/métodos , Osteogênese/efeitos dos fármacos , RNA Interferente Pequeno/uso terapêutico , Anabolizantes/administração & dosagem , Animais , Caseína Quinase II/antagonistas & inibidores , Feminino , Inativação Gênica/efeitos dos fármacos , Lipossomos/administração & dosagem , Lipossomos/uso terapêutico , Microscopia Confocal , Osteoblastos/efeitos dos fármacos , Osteoporose/tratamento farmacológico , RNA Interferente Pequeno/administração & dosagem , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real
3.
Nat Protoc ; 4(6): 984-91, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19498378

RESUMO

This protocol describes a simple, convenient and sensitive single-nucleotide polymorphism (SNP) genotyping method using an optically amplifying cationic conjugated polymer and single base primer extension reaction. The fluorescence resonance energy transfer (FRET) efficiency between the conjugated polymer (PFP, poly{(1,4-phenylene)-2,7-[9,9-bis(6'-N,N,N-trimethyl ammonium)-hexyl fluorene] dibromide}) and a fluorescein-labeled dNTP (dNTP-Fl) is correlated to the incorporation of the dNTP-Fl into an allele-specific primer; incorporation occurs by a single base extension reaction when the target DNA and the primer are complementary at the SNP site. By triggering the FRET from PFP to fluorescein and measuring the change in fluorescence intensity of samples, the SNP genotypes can be discriminated. In comparison with other SNP genotyping methods, this protocol simplifies procedures and improves sensitivity by eliminating the need for primer labeling, cumbersome workups, chemical/enzymatic coupling reactions and sophisticated instruments. The assay takes about 2 h for PCR amplification followed by 5.5-7.5 h to obtain the genotypes.


Assuntos
DNA/química , Genômica/métodos , Genótipo , Polímeros/química , Polimorfismo de Nucleotídeo Único , Cátions , Transferência Ressonante de Energia de Fluorescência/métodos , Reação em Cadeia da Polimerase/métodos
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