Aptamers against Cells Overexpressing Glypican 3 from Expanded Genetic Systems Combined with Cell Engineering and Laboratory Evolution

Abstract

Laboratory in vitro evolution (LIVE) might deliver DNA aptamers that bind proteins expressed on the surface of cells. Here, we use cell engineering to place on the surface of a liver cell line glypican 3 (GPC3), a possible marker for liver cancer diagnostics and theranostics. Libraries were then built from a six-letter genetic alphabet containing standard nucleobase and two added nucleobases (2-amino-8H-imidazo[1,2-a] [1,3,5] triazin-4-one and 6-amino-5-nitropyridin-2-one, trivially Z and P), Watson-Crick complements from an artificially expanded genetic information system (AEGIS). With counterselection against un-engineered cells, eight AEGIS-containing aptamers were recovered. Five bound selectively to GPC3 overexpressing cells. This selection-counterselection scheme had acceptable statistics, notwithstanding the possibility that cells engineered to overexpress GPC3 might also express different off-target proteins. This is the first example of such a combination.

Laboratory evolution of artificially expanded DNA gives redesignable aptamers that target the toxic form of anthrax protective antigen

“…This was not the first time that this was observed; other AEGIS GACT ZP LIVE experiments produced many successful aptamers containing only P residues. However, these experiments differed from these other experiments in that the latter experiments also produced many survivors that has one or more Z's (10–11,29). Further, a single sequence dominated the output, corresponding to ∼96% of the sequence reads.…”

Molecular Elucidation of Disease Biomarkers at the Interface of Chemistry and Biology

“…30 Another expanded genetic system used in SELEX was jointly reported by the Benner and Tan groups, in which a pair of artificial nucleotides was incorporated into the DNA library to select aptamers toward living cells 31,32 and cell surface targets. 110 ntroduction of this base pair not only added functionality to the library reservoir but also largely increased information density, providing much larger folding possibility of oligonucleotides, eventually making the entire selection process more efficient. These new generations of SELEX will increase in popularity and effectiveness when the related chemical biological technologies are fully equipped, especially the engineering of more effective polymerases to better accept artificial nucleobases and more efficient sequencing technologies for artificial nucleobase-containing DNA/RNA molecules.…”

Aptamer/AuNP Biosensor for Colorimetric Profiling of Exosomal Proteins

“…In particular, these “chemical antibodies” exhibit excellent binding affinity and specificity toward cell-surface proteins. [9] This characteristic of specific recognition has been harnessed as a versatile platform to develop biosensing and molecular imaging tools in the physiological environment. [10] Inspired by these achievements, we hypothesized the generation of a predictive signature-based strategy based on the binding between aptamer and exosome surface protein, which constitutes the basis for profiling the subtle variations in exosome protein patterns among different cell types to collect phenotype information of cancers.…”