Expansion of the Genetic Alphabet: A Chemist's Approach to Synthetic Biology

Abstract

The information available to any organism is encoded in a four nucleotide, two base pair genetic code. Since its earliest days, the field of synthetic biology has endeavored to impart organisms with novel attributes and functions, and perhaps the most fundamental approach to this goal is the creation of a fifth and sixth nucleotide that pair to form a third, unnatural base pair (UBP) and thus allow for the storage and retrieval of increased information. Achieving this goal, by definition, requires synthetic chemistry to create unnatural nucleotides and a medicinal chemistry-like approach to guide their optimization. With this perspective, almost 20 years ago we began designing unnatural nucleotides with the ultimate goal of developing UBPs that function in vivo, and thus serve as the foundation of semi-synthetic organisms (SSOs) capable of storing and retrieving increased information. From the beginning, our efforts focused on the development of nucleotides that bear predominantly hydrophobic nucleobases and thus that pair not based on the complementary hydrogen bonds that are so prominent among the natural base pairs but rather via hydrophobic and packing interactions. It was envisioned that such a pairing mechanism would provide a basal level of selectivity against pairing with natural nucleotides, which we expected would be the greatest challenge; however, this choice mandated starting with analogs that have little or no homology to their natural counterparts and that, perhaps not surprisingly, performed poorly. Progress toward their optimization was driven by the construction of structure-activity relationships, initially from in vitro steady-state kinetic analysis, then later from pre-steady-state and PCR-based assays, and ultimately from performance in vivo, with the results augmented three times with screens that explored combinations of the unnatural nucleotides that were too numerous to fully characterize individually. The structure-activity relationship data identified multiple features required by the UBP, and perhaps most prominent among them was a substituent ortho to the glycosidic linkage that is capable of both hydrophobic packing and hydrogen bonding, and nucleobases that stably stack with flanking natural nucleobases in lieu of the potentially more stabilizing stacking interactions afforded by cross strand intercalation. Most importantly, after the examination of hundreds of unnatural nucleotides and thousands of candidate UBPs, the efforts ultimately resulted in the identification of a family of UBPs that are well recognized by DNA polymerases when incorporated into DNA and that have been used to create SSOs that store and retrieve increased information. In addition to achieving a longstanding goal of synthetic biology, the results have important implications for our understanding of both the molecules and forces that can underlie biological processes, so long considered the purview of molecules benefiting from eons of evolution, and highlight the promise of applying the approaches and methodologies of synthetic and medical chemistry in the pursuit of synthetic biology.

Figure 1

dNaM-dTPT3, dZ-dP, and dDs-dPx (R = H or −CH(OH)–CH₂OH) UBPs.

Figure 2

(A) First generation methylated benzene and naphthalene analogs. (B) Second generation methylated benzene analogs. Sugar and phosphate groups omitted for clarity.

Figure 3

(A) Isocarbostyril analogs. (B) Heteroatom derivatized isocarbostyril analogs. (C) Furan and thiophene fused pyridone and thiopyridone analogs. Sugar and phosphate groups omitted for clarity.

Figure 4

Azaindole analogs. Sugar and phosphate groups omitted for clarity.

Figure 5

(A) Duplex structure of DNA containing the dPICS–dPICS UBP. (B) Duplex structure of DNA containing the dNaM–d5SICS UBP. (C) Structure of d5SICSTP paired opposite dNaM in the polymerase active site. In chemical structures, sugar and phosphate groups omitted for clarity.

Figure 6

(A) Bromo-, cyano-, and fluoro-substituted benzene analogs. (B) Methoxy-substituted benzene analogs. Sugar and phosphate groups omitted for clarity.

Figure 7

Derivatized monocyclic pyridone analogs. Sugar and phosphate groups omitted for clarity.

Figure 8

Pyridine and substituted pyridine analogs. Sugar and phosphate groups omitted for clarity.

Figure 9

(A) Para- and meta-substituted dMMO2 analogs. (B) Optimized dMMO2 analogs. Sugar and phosphate groups omitted for clarity.

Figure 10

(A) Superimposition of the binary complex of KlenTaq polymerase bound to DNA with dNaM in the templating position in the open conformation (yellow) and the corresponding ternary complex bound to d5SICSTP in the closed conformation (purple). (B) Superimposition of ternary complex between KlenTaq polymerase, dNaM template DNA, and d5SICSTP (purple), or a natural dG template and dCTP (gray). Reproduced from ref (38). Copyright 2012 Nature Publishing Group.

Figure 11

Distal ring-contracted d5SICS analogs. Sugar and phosphate groups omitted for clarity.

Figure 12

A family of well replicated dNaM–dTPT3-like UBPs. Sugar and phosphate groups omitted for clarity.

Figure 13

A family of UBPs optimized for in vivo expansion of the genetic alphabet. Sugar and phosphate groups omitted for clarity.