المرجع الالكتروني للمعلوماتية
المرجع الألكتروني للمعلوماتية
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Affinity Selection


  

2096       01:22 صباحاً       التاريخ: 30-11-2015              المصدر: M. E. Pennington, K. S. Lam, and A. E. Cress

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Affinity Selection
 
A key requirement of combinatorial library approaches is that desirable molecules must be segregated from the remaining library population. Segregation is most often accomplished by affinity partitioning, in which an immobilized target is used to capture interacting molecules from a solution-phase library. Target proteins can be immobilized either using antibodies bound to Staphylococcus protein A, using absorption of targets onto wells of plastic microtiter plates, or covalent attachment to a variety of polymeric supports. Alternatively, combinatorial libraries attached to solid supports—for example, one-bead, one-compound (OBOC) libraries—can be used to bind soluble targets. Solid-phase libraries have also been used to screen highly complex targets, such as living cells on which a variety of receptors are expressed (1). In all cases, unbound material is removed by washing, and the bound material is recovered for amplification or identification, depending on the nature of the library.
Plastic pins were used to develop the first peptide-based combinatorial libraries. The pins are
arranged such that they fit neatly into a single well of a microtiter plate. Thus, the pin system provides a convenient format for both synthesis and screening. The power of this method derives largely in the ease of handling large numbers of discrete syntheses in parallel. Sequential steps of the synthesis can be carried out by transferring the pin arrays through various microtiter reaction chambers. The peptides are then directly available for affinity screening, either on the solid phase or following cleavage from the solid support.
Lam et al. (2) developed another approach for peptide affinity selection involving split synthesis of peptides on solid support beads (OBOC libraries: see Combinatorial Synthesis). From a pool of millions of beads, a binding reaction is performed using a target molecule such as an antibody, receptor, enzyme, or even whole cells. Beads displaying affinity for the target are isolated, and the peptide on the bead can be microsequenced.
Fodor et al. (3) have developed immobilized combinatorial libraries on silicon microchips. Chip-based addressable libraries of peptides, oligonucleotides, and small organic molecules can be readily prepared. These methods use photolithography to control regions accessible for subsequent chemical modification. This method enables a miniaturized, fully addressable library to be generated on the surface of a silicon chip. The resulting arrays can be screened using standard affinity methods.
 The affinity selection methods described above are extremely broad in their applications. Although classical nucleic acid libraries have been screened by hybridization, which is a highly specialized type of affinity partitioning, modern DNA and RNA aptamer libraries are screened in identical fashion to other combinatorial libraries by affinity selection over immobilized targets. Although selection strategies have been introduced that are based on properties other than affinity, such methods are highly specialized and are not likely to displace the current reliance on affinity methods for combinatorial screening.
References
1. M. E. Pennington, K. S. Lam, and A. E. Cress (1996) Mol. Diversity 2, 19–28. 
2. K. S. Lam, S. E. Salmon, E. M. Hersh, V. J. Hruby, W. M. Kazmierski, and R. J. Knapp (1991) Nature 354, 82–84. 
3. S. P. Fodor, J. L. Read, M. C. Pirrung, L. Stryer, A. T. Lu, and D. Solas (1991) Science 251, 767–773.
 


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