Evolution of functional six-nucleotide DNA

Abstract

Axiomatically, the density of information stored in DNA, with just four nucleotides (GACT), is higher than in a binary code, but less than it might be if synthetic biologists succeed in adding independently replicating nucleotides to genetic systems. Such addition could also add functional groups not found in natural DNA, but useful for molecular performance. Here, we consider two new nucleotides (Z and P, 6-amino-5-nitro-3-(1'-β-D-2'-deoxyribo-furanosyl)-2(1H)-pyridone and 2-amino-8-(1'-β-D-2'-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one). These are designed to pair via complete Watson-Crick geometry. These were added to a library of oligonucleotides used in a laboratory in vitro evolution (LIVE) experiment; the GACTZP library was challenged to deliver molecules that bind selectively to liver cancer cells, but not to untransformed liver cells. Unlike in classical in vitro selection, low levels of mutation allow this system to evolve to create binding molecules not necessarily present in the original library. Over a dozen binding species were recovered. The best had Z and/or P in their sequences. Several had multiple, nearby, and adjacent Zs and Ps. Only the weaker binders contained no Z or P at all. This suggests that this system explored much of the sequence space available to this genetic system and that GACTZP libraries are richer reservoirs of functionality than standard libraries.

Figure 1

Crystal structures of C:G, T:A and Z:P pairs (top as structures, bottom space filling) showing their similarity (PDB ID: 4RHD): (A) C:G and T:A pairs. (B) Z:P pair retaining hydrogen bonding. The structure for this Z:P pair was obtained by co-crystallization of 5′-G^5-MeSedUGT-Z-ACAC-3′ and 5′-G^5-MeSedUGT-P-ACAC-3′, where Se derivatization was used to speed crystallization. For structures of unmodified DNA containing Z:P pairs, see [Georgiadis et al. submitted].

Figure 2

Schematic of the AEGIS cell-LIVE with both positive and counter selections.

Figure 3

DNA aptamers recovered from cell-LIVE. (A) Sequences (only showing randomized region), dissociation constants (Kd), and percent in pool of sequences of binders (the AEGIS Z and P are shown in red). Sequences are arranged in order of increasing dissociation constant. (B) Binding and specificity of selected DNA aptamers. Aptamers are arranged from the least tightly binding (top) to the most tightly binding (bottom). (left) Binding to transformed liver cells, the “positive” in the selection. (right) Binding to untransformed liver cells, the counter-selection cells. The red distributions at the bottom of each panel is the signal generated from DNA that has the same length as the aptamers but random sequence.

Figure 4

Binding of analogs of aptamer LZH3 and LZH7 with Z and P replaced by standard nucleotides. The indicated aptamers and its analogs (50 nM) were incubated with target cell (4 °C for 30 min), and then analyzed using flow cytometry as described for Figure 3