Biomedical Engineering Reference
In-Depth Information
was chemically modified with a trifunctional appendage (i.e., succinylaminohex-
anolsarcosine) comprising the following features: (1) an acid-labile cleavable site
for ensuring easy detachment of the DNA-encoded molecule after library synthesis,
(2) an
O
-DMT, and (3) an
N
-Fmoc moiety, to fulfill orthogonal chemistry require-
ments for the regiospecific elongation of the emerging coding oligonucleotide and
peptide sequence, respectively (Figure 11.4b) [48]. After releasing the final com-
pound from the solid support by acid cleavage, the correct peptide sequence was
confirmed by Edman sequencing and radiolabeled using a T4-polynucleotide kinase.
As anticipated, binding assays revealed the nanomolar affinity of the synthetic DNA-
tagged pentapeptide (Figure 11.4c) toward antileucine-enkephalin antibody (3-E7)
[49], thus indicating that the potential steric hindrance of the oligonucleotide tag does
not significantly affect the binding interaction [48]. Furthermore, the oligonucleotide
appendage could be efficaciously amplified and sequenced by means of conventional
polymerase chain reactions (PCRs), confirming the specificity and stability of the
coding sequence [48].
In the same year, Gallop and co-workers at Affymax, accomplished the synthesis
of the first DNA-encoded collection of small-molecule polypeptides, using a synthetic
strategy very similar to the one proposed by Brenner et al. (Figure 11.4) [47,50]. The
library was designed to contain 823,540 heptapeptide sequences (7
7
members). By
means of seven cycles of alternate split-pool synthesis, different D- and L-amino acid
building blocks were assembled in combinatorial fashion on small spherical beads
(Figure 11.4,
n
7). To trace the process by which each peptide sequence
had been assembled, dinucleotide DNA tags were introduced through a parallel
combinatorial procedure [50]. However, with the aim of avoiding depurination of
deoxyadenosine and deoxyguanosine during the trifluoroacetic acid cleavage of the
compounds from the support, an acid-resistant deoxyadenosine was used (c
7
dA) [51],
whereas deoxyguanosine nucleotides were deliberately excluded from the coding
oligonucleotides [50].
The final DNA-conjugated-heptapetide library was subjected to “on-bead” screen-
ing against a fluorescently labeled antipeptide antibody that selectively binds the
RQFKVVT peptide sequence. A fluorescence-activated sorting instrument (FACS)
allowed the isolation of those beads to which the antibody bound tightly. PCR ampli-
fication and DNA sequencing of the DNA tags attached to the sorted beads yielded
the identification of the expected antibody-specific heptapeptide (RQFKVVT) and
other analog sequences capable of binding the target with comparable affinities (
K
D
values ranging between 0.3 and 1400 nM) [50].
Even though these results marked the first milestone in the development of DNA-
encoded chemistry technology, the intrinsic limitations in terms of library size (which
could not be greater than the number of beads) and the need for a peculiar orthogo-
nal chemistry, combined with a cumbersome solid-phase DNA synthesis, seriously
hampered the evolution of this technology, which was never practically employed for
drug discovery purposes.
Research in the field of DNA encoding remained essentially unknown until the
end of the 1990s. Alternative chemical and physical strategies for the encoding of
small compounds had been pursued extensively with the aim of generating very large
libraries in a single test tube. Mass spectrometric-based tagging, peptide encoding,
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m
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