Biomedical Engineering Reference
In-Depth Information
cyclization. Reactions between precursors (i.e., oligomerization and polymerization)
become competitive and difficult to avoid [86]. Therefore, the principal method of
favoring the intramolecular cyclization consists in slowing the rate of intermolecu-
lar reactions by performing the macrocyclization at very low reagent concentration
(high dilution conditions) [86]. By contrast, in DNA-templated synthesis, the prox-
imity effect (see above) is concentration independent and enables the simultaneous
and specific assemble of thousands of macrocyclic variants in one pot. According to
such a principle, Ensemble Thera-peutics has generated several peptidic macrocycle
libraries, each containing up to 6
10
4
×
structures [81].
11.2.2.3.2 Chemetics
Nuevolution is a drug discovery company headquartered in
Copenhagen. Since 2001 it has developed several DNA-encoded chemical libraries
employing conventional split-pool techniques (see Section 11.2.2.1) as well as its
proprietary DNA-template-based technology (called
chemetic
). Although Nuevolu-
tion reported the use of chemetics in numerous patent applications on DNA-encoded
chemical library assembly, to date there is no peer-reviewed publication exhaustively
describing the methodology and how it is practically pursued for the construction of
DNA-encoded chemical libraries.
Such an approach seems to rely primarily on the control of the associa-
tion/dissociation equilibrium between the template and the reagent oligonucleotides
to modulate the DNA-templated reactivity, and thus library construction.
In a 2004 U.S. patent application, the Danish company presented the first exam-
ple of the chemetic strategy for library synthesis. In virtue of the proximity effect
(see above), random DNA sequences mediated the enzymatic incorporation (e.g., by
means of a polymerase) of unnatural DNA-nucleotide building blocks (each display-
ing two functional moieties and a cleavable linker) by a reaction between neighboring
reactive groups (Figure 11.17a) [87]. Eventually, the linker cleavage yielded the final
double-stranded DNA-encoded library (Figure 11.17a).
According to the authors, after library selection, the enriched DNA codons can be
PCR-amplified and used as molecular evolution templates (see Section 11.2.2.2) into
an additional round of library resynthesis and selection [87].
In a later patent, the construction strategy described before has been extended to
the use of partially complementary DNA-hairpin templates to prime the annealing
of short complementary DNA-conjugate reagents (Figure 11.17b) [88]. Following
ligation of DNA-linked reagent to the hairpin coding sequence, denaturation of the
duplex structure allows the DNA-templated reaction to occur (Figure 11.17b) [88].
In a more recent patent, Nuevolution described the latest example of chemetic
strategy for the assembly of DNA-encoded chemical libraries (called
zipper-box
approach
), as depicted schematically in Figure 11.17c [89]. Multiple DNA-conjugate
reagents, each containing two hybridization domains, are annealed consecutively
on an independent single-stranded DNA template (“zipper box”). According to the
patent application, it is possible to modulate the corresponding DNA-templated reac-
tion controlling the association/dissociation of two adjacent DNA-conjugate reagents
(e.g., performing PCR-like thermal cycles or using appropriate high/low salt concen-
tration buffers) [89]. If a bifunctional DNA-linked moiety is included, iteration of the
