Pool sequencing

In the rearrangement of divinylaziridines 289, the participation of a boat-like transition state 290 explained the stereochemical outcome of the reactions to give the azepinones 291 in 73 to 85% yield. The divinylaziridines 289 were synthesized via ex-chiral pool sequences starting from optically active a-amino acids. Table 16, Eq. (26) [55]. 

Third, we have selected aptamers that can bind to the 3II isozyme of protein kinase C from an RNA pool that spanned 120 random positions [5], The aptamers fell into several families, and individuals from two of the most prominent families were assayed for their ability to inhibit the enzymatic activity ofPKC. While these aptamers efficiently inhibited the enzymatic activity of the 3II isozyme, they had a 10-fold lower K, for the 3I isozyme (96% similar) and showed no activity against the a isozyme (80% similar). These specificities rival those seen with monoclonal antibodies. We have now selected aptamers that can bind to the a isozyme ofPKC from the same RNA pool. Sequence comparisons of the anti-pil and anti-a aptamers (Fig. 13) suggest that the map that relates target space and sequence space is convoluted. For example, while one family of aptamers was returned from both selections, other families were unique for one or the other isozyme. 

The term pool sequencing refers to the multiple sequence analysis through Edman degradation of peptide libraries composed of highly complex nnixtures. This analytical technique is valuable for the characterization of peptide hbraries in that it enables the qualitative and quantitative determination of each annino acid at each position of the sequence, thus, not only verifying equimolar representation of annino acids at each position, but also reconfirming completeness of couphng at each step. 

To synthesize highly diverse libraries, carrying more than six degenerated sequence positions (X), several methods using premixed mixtures of amino acids for the introduction of X positions in the sequence have been described [12]. Obviously, the frequency of each amino acid in a X position is influenced by the composition of the premixed amino acid mixture, the chemistry applied for coupling as well as by the nature of the solid support. The synthetic peptide libraries have been analyzed by electrospray mass spectrometry and pool sequencing [13,14]. Due to their stoichiometry, peptides from extremely diverse combinatorial collections are only partially accessible for biological assays [12]. 

Prominent primary anchor residues in anchor positions determined by pool sequencing (e.g., positions 5 and 8 for H-2Kb, positions 2 and 8 for H-2Ld) were ascertained by screening octapeptide libraries [39], Unfavorable effects on binding can be induced also by amino acids in non anchor positions. Conformational analysis of several individual peptides bound to MHC class I molecules showed that the position of the peptide backbone in the binding groove, as well as the orientation of side chains in other sequence positions, is influenced by individual amino acid side chains [36]. These interactions strongly determine the accessibility of the peptide on the MHC 1 surface and thus the response of the CTL effector cells. Anchor residues promote efficient binding to MHC 1, but notably suppress the CTL response to the complex peptide libraries [39]. 

The identity of the defined peptides was confirmed by electrospray mass spectrometry [13,62] and the purity of defined peptides was higher than 80% as determined by HPLC. The amino acid composition of selected peptide libraries and of the sublibraries was determined by pool sequencing [14] (Fig. 11.1) and amino acid analysis. Deviations from equimolar representation of the amino acids in randomized sequence positions were found to be within the error limits of the analytical methods [12]. Electrospray mass spectrometry for pattern analysis of peptide libraries differing in the position of the defined amino acid was carried out with a triple quadrupole mass spectrometer (Fisons, Manchester, UK) (Fig. 11.2). 

Figure 11.1. Results from pool sequencing (477 A, Applied Biosystems, Weiterstadt) of undeca- and tri-decapeptide libraries with one or four defined amino acid residues in different sequence positions. Bars indicate delected amounts of PTH-amino acids in each cycle. Edman degradation was carried out as described [14].

Slevanovic S, Jung G (1993) Multiple sequence analysis Pool sequencing of synthetic and natural peptide libraries, Anal Biochem 212 212-220. 

Falk K, Rotzschke O, Stevanovic S, Jung G, Rammensee HG (1994) Pool sequencing of natural HLA-DR, DQ and DP ligands reveals detailed peptide motifs, constraints of processing, and general rules, Immunogenetics 39 230-242. 

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