RESUMO
The assembly, stability, and timely disassembly of short interfering RNA (siRNA) nanocomplexes have the potential to affect the efficiency of siRNA delivery and gene silencing. As such, the design of new probes that can measure these properties without significantly perturbing the nanocomplexes or their environment may facilitate the study and further development of new siRNA nanocomplexes. Herein, we study Förster resonance energy transfer (FRET)-labeled siRNA probes that can track the assembly, stability, and disassembly of siRNA nanocomplexes in different environments. The probe is composed of two identical siRNAs, each labeled with a fluorophore. Upon nanocomplex formation, the siRNA-bound fluorophores become locally aggregated within the nanocomplex and undergo FRET. A key advantage of this technique is that the delivery vehicle (DV) need not be labeled, thus enabling the characterization of a large variety of nanocarriers, some of which may be difficult or even impossible to label. We demonstrate proof-of-concept by measuring the assembly of various DVs with siRNAs and show good agreement with gel electrophoresis experiments. As a consequence of not having to label the DV, we are able to determine nanocomplex biophysical parameters such as the extracellular apparent dissociation constants (K(D)) and intracellular disassembly half-life for several in-house and proprietary commercial DVs. Furthermore, the lack of DV modification allows for a true direct comparison between DVs as well as correlation between their biophysical properties and gene silencing.
Assuntos
Nanoestruturas/química , Sondas RNA/química , RNA Interferente Pequeno/química , Sequência de Bases , Sistemas de Liberação de Medicamentos , Transferência Ressonante de Energia de Fluorescência , Células HeLa , Humanos , Luciferases/genética , Nanotecnologia , Sondas RNA/administração & dosagem , Sondas RNA/genética , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , TransfecçãoRESUMO
U1snRNA, U3snRNA, 28 S ribosomal RNA, poly(A) RNA and a specific messenger RNA were visualized in living cells with microinjected fluorochrome-labeled 2' O-Methyl oligoribonucleotides (2' OMe RNA). Antisense 2' OMe RNA probes showed fast hybridization kinetics, whereas conventional oligodeoxyribonucleotide (DNA) probes did not. The nuclear distributions of the signals in living cells were similar to those found in fixed cells, indicating specific hybridization. Cytoplasmic ribosomal RNA, poly(A) RNA and mRNA could hardly be visualized, mainly due to a rapid entrapment of the injected probes in the nucleus. The performance of linear probes was compared with that of molecular beacons, which due to their structure should theoretically fluoresce only upon hybridization. No improvements were achieved however with the molecular beacons used in this study, suggesting opening of the beacons by mechanisms other than hybridization. The results show that linear 2' OMe RNA probes are well suited for RNA detection in living cells, and that these probes can be applied for dynamic studies of highly abundant nuclear RNA. Furthermore, it proved feasible to combine RNA detection with that of green fluorescent protein-labeled proteins in living cells. This was applied to show co-localization of RNA with proteins and should enable RNA-protein interaction studies.
Assuntos
Sondas RNA , RNA/metabolismo , Animais , Linhagem Celular , Proteínas Cromossômicas não Histona/genética , Citomegalovirus/genética , Corantes Fluorescentes/química , Proteínas de Fluorescência Verde , Humanos , Hibridização in Situ Fluorescente , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microinjeções , Microscopia de Fluorescência/métodos , Proteínas Nucleares/genética , Poli A/genética , Poli A/metabolismo , RNA/genética , Sondas RNA/administração & dosagem , Sondas RNA/química , Sondas RNA/genética , RNA Ribossômico 28S/genética , RNA Ribossômico 28S/metabolismo , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , RNA Nucleolar Pequeno/genética , RNA Nucleolar Pequeno/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Proteínas de Ligação a RNA , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Processamento de Serina-Arginina , Células Tumorais CultivadasRESUMO
A localizaçao celular do pró-colágeno 1(I) foi observada na interface de coral natural, sete dias após implante no nosso trabecular de ratos, utilizando-se sondas de RNA marcadas com digoxigenina. A eficácia deste método foi comparada com aquela de sondas de RNA marcadas com enxofre(35). A hibridizaçäo "in situ" realizada utilizando-se sondas de RNA de osteoblastos, marcada com digoxigenina, provou ser täo eficiente quanto aquela marcada com S(35). Embora a porcentagem de fibroblastos marcados com S(35) tenha sido maior do que aqueles marcados com digoxigenina, näo houve diferença significante quanto à eficiência dos dois métodos
