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. 2019 Jul 10;141(27):10644-10653.
doi: 10.1021/jacs.9b02075. Epub 2019 Jun 26.

Optimization of Replication, Transcription, and Translation in a Semi-Synthetic Organism

Aaron W Feldman  1 , Vivian T Dien  1 , Rebekah J Karadeema  1 , Emil C Fischer  1 , Yanbo You  2 , Brooke A Anderson  1 , Ramanarayanan Krishnamurthy  1 , Jason S Chen  1 , Lingjun Li  2 , Floyd E Romesberg  1
Affiliations

Optimization of Replication, Transcription, and Translation in a Semi-Synthetic Organism

Aaron W Feldman et al. J Am Chem Soc. .

Abstract

Previously, we reported the creation of a semi-synthetic organism (SSO) that stores and retrieves increased information by virtue of stably maintaining an unnatural base pair (UBP) in its DNA, transcribing the corresponding unnatural nucleotides into the codons and anticodons of mRNAs and tRNAs, and then using them to produce proteins containing noncanonical amino acids (ncAAs). Here we report a systematic extension of the effort to optimize the SSO by exploring a variety of deoxy- and ribonucleotide analogues. Importantly, this includes the first in vivo structure-activity relationship (SAR) analysis of unnatural ribonucleoside triphosphates. Similarities and differences between how DNA and RNA polymerases recognize the unnatural nucleotides were observed, and remarkably, we found that a wide variety of unnatural ribonucleotides can be efficiently transcribed into RNA and then productively and selectively paired at the ribosome to mediate the synthesis of proteins with ncAAs. The results extend previous studies, demonstrating that nucleotides bearing no significant structural or functional homology to the natural nucleotides can be efficiently and selectively paired during replication, to include each step of the entire process of information storage and retrieval. From a practical perspective, the results identify the most optimal UBP for replication and transcription, as well as the most optimal unnatural ribonucleoside triphosphates for transcription and translation. The optimized SSO is now, for the first time, able to efficiently produce proteins containing multiple, proximal ncAAs.

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Conflict of interest statement

Notes

The authors declare the following competing financial interests: a patent application has been filed based on the use of UBPs in SSOs and F.E.R. has a financial interest (shares) in Synthorx, Inc., a company that has commercial interests in the UBP.

Figures

Figure 1.
Figure 1.
The dNaM-d5SICS, dNaM-dTPT3, dCNMO-dTPT3, and dPTMO-dTPT3 UBPs.
Figure 2.
Figure 2.
dXTP analogs. Ribose and phosphates omitted for clarity.
Figure 3.
Figure 3.
Optimization of translation to incorporate AzK into sfGFP using various dXTPs. (A) UBP retention (%) in the sfGFP gene. (B) UBP retention (%) in the tRNAPyl gene. (C) Relative sfGFP fluorescence observed normalized to cell growth (Relative Fluorescence Units (RFU) per OD600) in the presence or absence of AzK. (D) Protein shift (%) measured by western blot. Each bar represents the mean, with error bars indicating standard error (n = 3). Open circles represent data for each independent trial. Asterisk indicates that cells were unable to grow under the condition indicated.
Figure 4.
Figure 4.
Ribonucleotide analogs. (A) XTP analogs. (B) YTP analogs. Ribose and phosphates omitted for clarity.
Figure 5.
Figure 5.
SAR analysis of translation using various unnatural ribonucleotides to incorporate AzK into sfGFP. (A) Total sfGFP fluorescence (RFU) observed in the presence of AzK for XTP analogs, where (−) represents control samples provided with only TPT3TP (XTP withheld). (B) Protein shift (%) measured by western blot for XTP analogs, where (−) represents control samples provided with only TPT3TP (XTP withheld). (C) Total sfGFP fluorescence (RFU) observed in the presence of AzK for YTP analogs, where (−) represents control samples provided with only NaMTP (YTP withheld). (D) Protein shift (%) measured by western blot for YTP analogs, where (−) represents control samples provided with only NaMTP (YTP withheld). Each bar represents the mean, with error bars indicating standard error (n = 4). Open circles represent data for each independent trial.
Figure 6.
Figure 6.
Optimization of unnatural ribonucleotide triphosphate concentrations. (A) Total sfGFP fluorescence (RFU) as a function of the concentrations of NaMTP and TAT1TP (µM). (B) Total sfGFP fluorescence (RFU) as a function of the concentrations of 5FMTP and TAT1TP (µM). (C) Protein shift (%) as a function of the concentrations of NaMTP and TAT1TP (µM). (D) Protein shift (%) as a function of the concentrations of 5FMTP and TAT1TP (µM). Error bars indicate standard error of each value (n = 3).
Figure 7.
Figure 7.
Storage and retrieval of higher density unnatural information with either dNaM-dTPT3/NaMTP,TPT3TP or dCNMO-dTPT3/NaMTP,TAT1TP. (A) Total sfGFP fluorescence (RFU) observed in the presence of AzK. (B) Protein shift (%) measured by western blot. For strip charts, each bar represents the mean, with error bars indicating standard error (n = 4), and open circles represent data for each independent trial. (C) Representative spectrum of quantitative HRMS analysis of triple labeled protein produced using the dCNMO-dTPT3/NaMTP,TAT1TP. Peak labels show deconvoluted molecular weight of intact protein, with amino acid residues at positions 149, 151, and 153 shown and quantification of each peak (%, n = 3) shown below. See Supporting Information for assignment of unlabeled peaks.
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