Expanding the Scope of Replicable Unnatural DNA: Stepwise Optimization of a Predominantly Hydrophobic Base Pair

Abstract

As part of an ongoing effort to expand the genetic alphabet for in vitro and eventually in vivo applications, we have synthesized a wide variety of predominantly hydrophobic unnatural base pairs exemplified by d5SICS-dMMO2 and d5SICS-dNaM. When incorporated into DNA, the latter is replicated and transcribed with greater efficiency and fidelity than the former, however previous optimization efforts identified the para and methoxy-distal meta positions of dMMO2 as particularly promising for further optimization. Here, we report the stepwise optimization of dMMO2 via the synthesis and evaluation of eighteen novel para-derivatized analogs of dMMO2, followed by further derivatization and evaluation of the most promising analogs with meta substituents. Subject to size constraints, we find that para substituents can optimize replication via both steric and electronic effects and that meta methoxy groups are unfavorable while fluoro substituents can be beneficial or deleterious depending on the para substituent. In addition, we find that improvements in the efficiency of unnatural triphosphate insertion translate most directly into higher fidelity replication. Importantly, we identify multiple, unique base pair derivatives that when incorporated into DNA are well replicated. The most promising, d5SICS-dFEMO, is replicated under some conditions with greater efficiency and fidelity than d5SICS-dNaM. These results clearly demonstrate the generality of hydrophobic forces for the control of base pairing within DNA, provide a wealth of new SAR data, and importantly identify multiple new candidates for eventual in vivo evaluation.

Fluorescent Nucleobase Analogues with Extended Pi Surfaces Stabilize DNA Duplexes Containing O6‐Alkylguanine Adducts

“…This result is consistent with previous observations for Per and may be rationalized on the basis of hydrophobic interactions favouring probe pairing with the more hydrophobic O 6 ‐alkylG adducts than with polar canonical nucleobases. This hydrophobic pairing phenomenon has been previously observed with other non‐natural nucleosides . The highest DNA duplex stabilities were observed for ExBenzi paired with either of the O 6 ‐alkylG adducts, while similar stabilization of O 6 ‐alkylG was observed for both ExBIM and Per ( Figure , Table ).…”

In Vivo Structure–Activity Relationships and Optimization of an Unnatural Base Pair for Replication in a Semi-Synthetic Organism

“…In fact, d CNMO -d TPT3 shows at least moderate retention in sequence context 4 , where d NaM -d TPT3 is retained so poorly that it cannot be rescued by Cas9, suggesting that it is lost immediately upon attempted replication. Interestingly, this contrasts with in vitro data where retention of d NaM -d TPT3 is better than retention of d CNMO -d TPT3 , 8,15 suggesting that E. coli provide a unique environment for which d CNMO -d TPT3 is more optimal. Possible contributing factors include Pt NTT2-mediated uptake, stability within the cell, or recognition by different polymerases that can access the replication fork and actually mediate replication in vivo .…”

The Expanded Genetic Alphabet

“…After efforts to further optimize d NaM resulted in only flat SAR, [134,138-140] our efforts turned to the optimization of d 5SICS , since continued primer extension after the incorporation of this unnatural nucleotide limits replication. These efforts eventually yielded d TPT3 (Figure 4).…”