RESUMO
A number of aptamers have been selected against cell surface biomarkers or against eukaryotic tissue culture cells themselves. To determine the general utility of aptamers for assessing the cell surface proteome, we developed a standardized flow cytometry assay and carried out a comprehensive study with 7 different aptamers and 14 different cell lines. By examining how aptamers performed with a variety of cell lines, we identified difficulties in using aptamers for cell typing. While there are some aptamers that show excellent correlation between cell surface binding and the expression of a biomarker on the cell surface, other aptamers showed nonspecific binding by flow cytometry. For example, it has recently been claimed that an anti-PTK7 (protein tyrosine kinase 7) aptamer identified a new biomarker for leukemia cells, but data with the additional cell lines shows that it is possible that the aptamer instead identifies a propensity for adherence. Better understanding and controlling for the role of background and nonspecific binding to cells should open the way to using arrays of aptamers for describing and quantifying the cell surface proteome.
Assuntos
Aptâmeros de Nucleotídeos/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Membrana/metabolismo , Proteômica/métodos , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Sequência de Bases , Western Blotting , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Citometria de Fluxo , Humanos , Proteínas de Membrana/genética , Microscopia Confocal , Modelos Biológicos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Ligação Proteica , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismoRESUMO
In vitro selection experiments show first and foremost that it is possible that functional nucleic acids can arise from random sequence libraries. Indeed, even simple sequence and structural motifs can prove to be robust binding species and catalysts, indicating that it may have been possible to transition from even the earliest self-replicators to a nascent, RNA-catalyzed metabolism. Because of the diversity of aptamers and ribozymes that can be selected, it is possible to construct a 'fossil record' of the evolution of the RNA world, with in vitro selected catalysts filling in as doppelgangers for molecules long gone. In this way a plausible pathway from simple oligonucleotide replicators to genomic polymerases can be imagined, as can a pathway from basal ribozyme activities to the ribosome. Most importantly, though, in vitro selection experiments can give a true and quantitative idea of the likelihood that these scenarios could have played out in the RNA world. Simple binding species and catalysts could have evolved into other structures and functions. As replicating sequences grew longer, new, more complex functions or faster catalytic activities could have been accessed. Some activities may have been isolated in sequence space, but others could have been approached along large, interconnected neutral networks. As the number, type, and length of ribozymes increased, RNA genomes would have evolved and eventually there would have been no area in a fitness landscape that would have been inaccessible. Self-replication would have inexorably led to life.
Assuntos
Evolução Molecular Direcionada , Evolução Molecular , RNA/genética , Animais , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Sequência de Bases , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA/química , RNA Catalítico/química , RNA Catalítico/genéticaRESUMO
Drug-resistant variants of HIV-1 reverse transcriptase (RT) are also known to be resistant to anti-RT RNA aptamers. In order to be able to develop diagnostics and therapies that can focus on otherwise drug-resistant viruses, we have isolated two aptamers against a well-known, drug-resistant HIV-1 RT, Mutant 3 (M3) from the multidrug-resistant HIV-1 RT panel. One aptamer, M302, bound M3 but showed no significant affinity for wild-type (WT) HIV-1 RT, while another aptamer, 12.01, bound to both M3 and WT HIV-1 RTs. In contrast to all previously selected anti-RT aptamers, neither of these aptamers showed observable inhibition of either polymerase or RNase H activities. Aptamers M302 and 12.01 competed with one another for binding to M3, but they did not compete with a pseudoknot aptamer for binding to the template/primer cleft of WT HIV-1 RT. These results represent the surprising identification of an additional RNA-binding epitope on the surface of HIV-1 RT. M3 and WT HIV-1 RTs could be distinguished using an aptamer-based microarray. By probing protein conformation as a correlate to drug resistance we introduce an additional and useful measure for determining HIV-1 drug resistance.