Biomedical Engineering Reference
In-Depth Information
anticodon columns for further chemical modifications. Such a split-pool process can
be iterated for all codon regions until all the desired building blocks are incorporated
on the initial DNA templates.
In a first practical implementation of the DNA-routed strategy (also termed
DNA
display
), a double-stranded library containing 10
6
different 340-mer-oligonucleotide
templates was generated by combinatorial PCR assembly of smaller oligonucleotides
(Figure 11.12) [59]. Following the conversion of the DNA-duplex format into a
single strand (by reverse transcription and base-mediated hydrolysis of the RNA
strand), the oligonucleotide library was chemically translated into 10
6
N
-acylated
pentapeptides using 10 different Fmoc-protected amino acids and nine carboxylic
acids for the terminal
N
-acylation (
N
-terminus unmodified derivatives were also
included) (Figure 11.12). Five of the six coding regions (20 bases each) present
on the initial oligonucleotide template allowed for unambiguous encoding of the
pentapeptidic sequences, while the sixth coding region ensured the identity of the
carboxylic acids used for the final
N
-acylation (Figure 11.12).
Acylated leucine-enkephalin pentapetides were intentionally included as positive
controls during library synthesis [59]. The library sequencing before and after two
rounds of selection against a single-digit nanomolar leucine-enkephalin antibody
(the same as that used by Brenner et al., in model selection experiments) [48,49]
revealed a remarkable round-to-round enrichment of the leucine-enkephalin consen-
sus sequences [59]. Notably, the eluted DNA from each step was PCR-amplified
and served as a evolutionary input for the subsequent round of chemical transla-
tion and selection. In this regard, the work of Halpin and Harbury represents the first
experimental implementation of molecular evolution using a DNA-encoded chemical
library [48,59,70].
More recently, Wrenn et al. applied DNA routing to the chemical translation of
an octamer peptoid library, comprising approximately 100 million compounds [71].
Six rounds of evolutionary selection against
N
-CrkSH3 (
N
-terminal SH3 domain of
the proto-oncogene Crk protein) yielded to the identification of peptoids that after
resynthesis revealed binding affinity ranging between
K
D
=
M [71].
Although at first glance DNA-routing strategies appear to be cumbersome and
tedious, the possibility of chemical translation and diversification holds promises
for the construction of large libraries whose sizes increase exponentially after each
round of synthesis and encoding. On the other hand, from a drug discovery point of
view, the simple polypeptidic structures which have been assembled so far using such
technology may not represent the drug-like structures in which the pharmaceutical
industry is traditionally interested in [68].
16 and 97
11.2.2.3 DNA-Directed Libraries
The ability of a DNA double helix to direct
region-specific chemical reactions by bringing DNA-linked reagents in close prox-
imity through Watson-Crick base pairing has long been known [72,73]. But it wasn't
until 2001 that Gartner and Liu introduced the concept of DNA-templated synthesis
(DTS) to show that such a proximity effect is a general strategy to locally increase
the effective molarity and thus the reaction rates [74].
