Systematic exploration of a class of hydrophobic unnatural base pairs yields multiple new candidates for the expansion of the genetic alphabet

Abstract

We have developed a family of unnatural base pairs (UBPs), which rely on hydrophobic and packing interactions for pairing and which are well replicated and transcribed. While the pair formed between d5SICS and dNaM (d5SICS-dNaM) has received the most attention, and has been used to expand the genetic alphabet of a living organism, recent efforts have identified dTPT3-dNaM, which is replicated with even higher fidelity. These efforts also resulted in more UBPs than could be independently analyzed, and thus we now report a PCR-based screen to identify the most promising. While we found that dTPT3-dNaM is generally the most promising UBP, we identified several others that are replicated nearly as well and significantly better than d5SICS-dNaM, and are thus viable candidates for the expansion of the genetic alphabet of a living organism. Moreover, the results suggest that continued optimization should be possible, and that the putatively essential hydrogen-bond acceptor at the position ortho to the glycosidic linkage may not be required. These results clearly demonstrate the generality of hydrophobic forces for the control of base pairing within DNA, provide a wealth of new structure–activity relationship data and importantly identify multiple new candidates for in vivo evaluation and further optimization.

A semisynthetic organism engineered for the stable expansion of the genetic alphabet

“…1A) being a particularly promising example (5)(6)(7). Despite lacking complementary hydrogen bonding, we demonstrated that the dNaM-d5SICS UBP is well replicated by a variety of DNA polymerases in vitro (7)(8)(9)(10), and that this efficient replication is mediated by a unique mechanism that draws upon interbase hydrophobic and packing interactions (11,12).…”

Evolvability Is an Evolved Ability: The Coding Concept as the Arch-Unit of Natural Selection

“…Apart from the continuing quest for examples of life outside Earth, investigations of possible shadow biospheres hidden on our planet (Davies 2011), and diverse mathematical models of evolvability under different conditions and constraints, informed by extrapolations of present physical knowledge and/or information-theoretical considerations (Adami 2015), the phenomenon of evolvable evolvability by natural selection at the level of the coding concept could also be experimentally tested in biological laboratories, by looking at evolvability of differently conceived life-forms. This may be accomplished imminently, using, for example, semi-synthetic organisms with different genetic alphabet Dhami et al 2014;Cleaves et al 2015) or, alternatively, semi-synthetic (or even fully synthetic) organisms with differentially designed systems for protein synthesis (Daube and Bar-Ziv 2013). Of note, the former could be a tool to test the evolvability-associated effects of different semantics, while the latter would enable us to test variations of syntax.…”

Unnatural Base Pairs for Synthetic Biology

“…Consequently, they explored more than 100 kinds of unnatural base pairs [19][20][21][22][23][24][25] and succeeded in developing 5SICS : MMO2 and 5SICS : NaM pairs, which are replicable unnatural base pairs in the PCR. [26][27][28] In 2014, they reported the creation of a semi-synthetic organism containing the 5SICS : NaM base pair. In this work, an exogenously expressed nucleoside triphosphate transporter imported d5SICS and dNaM triphosphates efficiently into E. coli, and an endogenous replication system used them in the genetic codes.…”

“…7d). Although the replication fidelity of the ImN N : NaO O pair is slightly inferior to those of other unnatural base-pair analogs, 8,9,26,28,34,46,47) it is strongly indicated that the ImN N : NaO O pair acts as an orthogonal base pair for WC base pairs during PCR amplification.…”