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【bio-news】Nucleic acid-based techniques for post-transcri
Decoy RNAs, also known as aptamers, are small RNAs designed to bind with high affinity to target molecules and inhibit function through steric hindrance and interference with interacting molecules [6..]. Two groups have now developed a high-throughput technique to screen a random RNA library for candidate aptamer ligation to target proteins [6..]. Using this selection process, Ostendorf et al. [34] developed an aptamer against the platelet-derived growth factor B isoform, which they utilized in rats with anti-Thy-1.1-induced mesangial proliferative glomerulonephritis. Aptamer treatment ameliorated both acute and chronic sequelae of nephritis, as determined by histopathology and proteinuria. In this instance, the aptamer strategy was critical in defining a role for platelet-derived growth factor in glomerulonephritis pathogenesis. This same group published an earlier report [35] using an aptamer against a VEGF receptor in the same model of glomerular disease. This aptamer was specific for endothelial cell effects, including inhibition of corneal angiogenesis. In renal disease studies, VEGF receptor Table 1. Comparison between nucleic acid methods for target molecule modulation Method Advantages Disadvantages Ribozymes Established methods with recognized mechanisms of activitySpecificity of activity Ability to delete or modify target mRNA Technically difficult and expensive to synthesize Large construct size can make cell delivery problematic Antisense oligonucleotides Established methods, with multiple modifications over
years to enhance efficacy Simple synthesis methods Lack of specificity due to non-sequence-dependent effects can be a problem Inconsistent target inhibition; may require trials with many constructs Aptamers Relatively simple synthesis methods Specificity of activityAvailability of methods to screen aptamer libraries New technology, with very limited experience RNA interference Relatively simple synthesis methods Specificity of activity Ability to produce siRNA stable transfectants New technology, with limited, but rapidly growing experience Inconsistent target inhibition; may require trials with many
constructs mRNA, messenger RNA; siRNA, small interfering RNA. Post-transcriptional regulation techniques Jarad et al. 417 inhibition by the aptamer was associated with decreased proliferation and increased apoptosis of glomerular endothelial cells, as well as increased proteinuria. Because these findings were restricted to endothelial cells and no aptamer effect was observed on two other models of glomerular disease, the authors concluded that recovery from anti-Thy-1.1 antibody-induced experimental glomerulonephritis was dependent upon endothelial cell proliferation. RNA interference
RNAi is a process by which double-stranded, 21- nucleotide, siRNAs post-transcriptionally inhibit gene expression by targeting homologous mRNA for degradation. RNAi is speculated to be an evolutionarily conserved mechanism for host plant and animal cells to defend against foreign or invading nucleic acids, such as transposons, viruses or aberrant mRNA, thereby functioning as a genomic immune system [36]. Several labs recently made the startling discovery that RNAi also plays a critical role in heterochromatin regulation required for Saccharomyces pombe and Tetrahymena spp. mitosis [37–39].
The biochemical pathways of RNAi are still being investigated [36,40.] (Fig. 2), but fundamental steps include endogenous double-stranded RNA (dsRNA) incorporation into an RNA–protein complex that includes a helicase, a kinase and a type III ribonuclease, DICER, which processes long dsRNAs into 21–25-nucleotide, double-stranded siRNA with a two to three nucleotide 3’-overhang. Although DICER appears to be important for RNAi in lower organisms, it has not been detected in mammalian systems, which explains the non-specific interferon inductionand cell death in mammalian cells treated with siRNA constructs longer than 30 base pairs. Helicase-unwound single-stranded RNA becomes part of the larger RNA-induced silencing complex and guides the complex to target mRNA. The bound siRNA/mRNA duplex is finally cut by an RNA-induced silencing
complex endoribonuclease, thereby inactivating translation. Although many investigators are continuing to search for fundamental roles of naturally occurring siRNAs, the
value of RNAi as an investigative tool for in-vitro cell and molecular biology experiments has grown enormously since the initial description of RNAi in mammalian systems [41]. Although we are unaware of head-to-head comparisons between efficacy of siRNAs and antisense oligonucleotides, because RNAi appears to be a specific and natural mechanism of mRNA silencing, we speculate that it may be a preferable approach to inhibition with antisense oligonucleotides (Table 1).
As an example of RNAi application to kidney biology investigation, we used the technique to define the role of a proximal tubule epithelial cell integrin [42]. To identify pathways regulated by the Fas death receptor, we initially used complementary DNA microarrays to demonstrate that b8 integrin subunit expression was induced in Fas-stimulated proximal tubule cells. To determine if the integrin was functionally regulated by Fas, we tested whether Fas induction of b8 expression correlated with migration to vitronectin, the only known ligand for b8 integrin at that time. Because function-blocking b8 integrin antibodies were not available, we instead developed negative controls by targeting b8 integrin using RNAi according to established methods for siRNA construct synthesis and transient transfection in mammalian systems (website:
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作者:admin@医学,生命科学 2011-06-21 05:11
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