In vitro selection with artificial expanded genetic information systems

Abstract

Artificially expanded genetic information systems (AEGISs) are unnatural forms of DNA that increase the number of independently replicating nucleotide building blocks. To do this, AEGIS pairs are joined by different arrangements of hydrogen bond donor and acceptor groups, all while retaining their Watson-Crick geometries. We report here a unique case where AEGIS DNA has been used to execute a systematic evolution of ligands by exponential enrichment (SELEX) experiment. This AEGIS-SELEX was designed to create AEGIS oligonucleotides that bind to a line of breast cancer cells. AEGIS-SELEX delivered an AEGIS aptamer (ZAP-2012) built from six different kinds of nucleotides (the standard G, A, C, and T, and the AEGIS nonstandard P and Z nucleotides, the last having a nitro functionality not found in standard DNA). ZAP-2012 has a dissociation constant of 30 nM against these cells. The affinity is diminished or lost when Z or P (or both) is replaced by standard nucleotides and compares well with affinities of standard GACT aptamers selected against cell lines using standard SELEX. The success of AEGIS-SELEX relies on various innovations, including (i) the ability to synthesize GACTZP libraries, (ii) polymerases that PCR amplify GACTZP DNA with little loss of the AEGIS nonstandard nucleotides, and (iii) technologies to deep sequence GACTZP DNA survivors. These results take the next step toward expanding the power and utility of SELEX and offer an AEGIS-SELEX that could possibly generate receptors, ligands, and catalysts having sequence diversities nearer to that displayed by proteins.

Keywords: cancer cell SELEX; next generation sequencing; nucleic acids; synthetic biology.

Fig. 1.

(Left) Watson–Crick pairing follows two rules of complementarity: (i) size complementarity (large purines pair with small pyrimidines) and (ii) hydrogen bonding complementarity [hydrogen bond donors (blue) pair with hydrogen bond acceptors (red)]. Rearranging hydrogen bond donors and acceptors on the bases gives an artificially expanded genetic information system (AEGIS). An xNA biopolymer having functionalized AEGIS components may allow SELEX to yield protein-like aptamers better than the standard DNA and RNA biopolymers. The example of AEGIS–SELEX reported here adds the Z:P pair, shown at the bottom of the molecular structures. (Right) The images show the general idea behind AEGIS in cartoon form.

Fig. 2.

Schematic of the AEGIS cell–SELEX used here. A GACTZP DNA library, consisting of randomized sequences and primer sites, was first incubated with the target cells. Unbound sequences were then removed by washing. Bound sequences having affinity to the target cells were collected. “Survivors” in the enriched collection were amplified using a FITC-labeled 5′-primer and a biotinylated 3′-primer by GACTZP six-nucleotide PCR (22). Enriched FITC-conjugated single-stranded DNA obtained from PCR products was used for the next round of selection. The binding affinity of survivors from 9th round up to 12th round of selection was monitored by flow cytometer. The survivors from 12th round selection were subjected to deep sequencing.

Fig. 3.

Monitoring the progress of GACTZP AEGIS Cell–SELEX using flow cytometer. Vertical axis indicates the number of cells counted having the fluorescence intensity indicated by the horizontal axis, with the increase in intensity from 9th round indicating the increased number of fluorescein-labeled aptamers bound per cell. Binding assays showed the enrichment of breast cancer cell (MDA-MB-231) binders with an optimum obtained after 11th round, and no further improvement after the 12th round.

Fig. 4.

Flow cytometry measurement of the binding of chemically resynthesized ZAP-2012 aptamer (5′-biotin-TCCCGAGTGACGCAGC-CCCCGGZGGGATTPATCGGT-GGACACGGTGGCTGAC-3′, the random region is underlined) obtained using AEGIS Cell–SELEX to MDA-MB-231 breast cancer cells (green curve). The vertical axis indicates the number of cells counted having the fluorescence (from biotin-bound Cy5.5-conjugated streptavidin) intensity indicated by the horizontal axis. Also shown (other colors) are binding data for chemically resynthesized ZAP-2012 mutants. (Upper) Where the Z and P are separately replaced with standard nucleotides, binding affinity is reduced. (Lower) Where the Z and P are both replaced with standard nucleotides, binding affinity is lost.

Fig. 5.

Estimation of the dissociation constant (K_diss) for the ZAP-2012 aptamer. (Upper) Binding signals of diluted aptamers (0.1–500 nM). (Lower) Binding curve, K_diss = 30 ± 1 nM.