Biological Function-Based Assays

As measured by the CD-based assay (Figure 1) the activity relative to WT varied from 70% (I78M) to 106% (L133M) for the single methionine mutants (Table II). Despite alteration of the core composition, the ten methionine variant retained activity equal to about 20% that of wild-type. The activity measurements therefore suggest that the single methionine substitutions described here have a small effect on biological function. 

Recently, Schreiber et al. have used macrolides and their linear precursors to probe the relative influences of stereochemical and skeletal diversity upon biological function. A library of 122 macrolides and their 122 linear precursors (Figure 9.5d) was evaluated in 40 different cell-based assays. Statistical analysis of the results revealed... 

Some RNA aptamers that were isolated in vitro have also been expressed in vivo to study their biological function within the context of a living pro- or eucaryotic cell. Among them is an aptamer which binds to the reverse transcriptase (Rev) protein of the human immunodeficiency virus type 1 (HIV-1) [42,43,70],This anti-HIV-l-Rev aptamer was cloned into an expression cassette based on the U6 snRNA promoter, in which aptamer transcripts are protected against nuclease degradation to some extent. Transient expression in the nucleus of cultured cells led to 107-109 full-length aptamer transcripts per cell. When anti-HIV-l-Rev aptamer-expressing cells were co-transfected with the HIV-1 provirus, viral replication was efficiently inhibited in these cells, as shown by an assay in which the production of HIV-1 reverse transcriptase was measured [71],... 

In contrast to the CLB assay format, cell-based bioassays are more complex, time, consuming, costly, and often are less sensitive. However, it is important to note that this methodology is a functional assay and, thus, demonstrates neutralization of a biological response. It is argued by some that a functional end point more closely mimics in vivo physiological responses [15]. 

The design of new proteins that can assemble into filaments, symmetric cages, and regular assays was described by Yeates and Padilla.These investigations are based on the consideration that proteins self-assemble to fulfill their biological functions and also as part of a... 

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