Selective Enhancement of Ligands by Exponential Enrichment (SELEX) has proven to be a powerful tool for idenfying functional oligonucleotides which bind to selected targets. Here, in an iterative process, a pool of single stranded oligonucleotides is contacted with a target structure (e.g. a protein), high-affinity oligonucleotides are then isolated, amplified and again put into contact with the target until, eventually, an ideal oligonucleotide has been obtained.
SELEX has opened up new avenues e.g. for generating aptamers as therapeutic compounds - and thereby enlarging the space for novel lead structures. However, the chemical space and thus the number of targetable proteins is still restricted by the limited diversity of nucleic acids. Use of chemically modified nucleic acids can solve this issue, but such nucleotides often lack compatibility with enzymatic steps (e.g. PCR) of the in vitro selection process.
Here, a versatile method (Click-SELEX) has been developed for the modular expansion of the chemical space of nucleic acid libraries, thus enabling the generation of nucleobase-modified aptamers with unprecedented recognition properties. The introduction of additional chemical entities into the DNA is achieved by using a 5’ modified, commercially available nucleoside-triphosphate whereas the modification can be further derivatized with so-called bio-orthogonal chemistry, e.g. click-chemistry.
Advantageously, the modification is only transiently present during the selection cycle. This enable a high degree of compatibility with the enzymatic step of the selection process. This approach further allows introducing larger chemical entities into DNA libraries that cannot be used using current methods.
The click chemistry has a broad acceptance of diverse substrates, which are either commercially available or accessible by chemical synthesis.
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Mayer, G., et al. (2016) Click reaction on solid phase enables high fidelity synthesis of nucleobase-modified DNA. Bioconjug. Chem. Feb 5., epub ahead of print.
Dellafiore, M.A., et al. (2016) Modified nucleoside triphosphates for in-vitro selection techniques. Front. Chem. 4: 19, epub.
Tolle, F., et al. (2015) A versatile approach towards nucleobase-modified aptamers. Angew. Chem. Int. Ed. Engl. 54(37): 10971-4.