Natural-like Replication of an Unnatural Base Pair for the Expansion of the Genetic Alphabet and Biotechnology Applications

Li, Lingjun; Degardin, Mélissa; Lavergne, Thomas; Malyshev, Denis A.; Dhami, Kirandeep; Ordoukhanian, Phillip; Romesberg, Floyd E.

doi:10.1021/ja408814g

Cited by 142 publications

(162 citation statements)

References 32 publications

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“…However, since the development of DM1 with the dNaM-d5SICS UBP, we have determined that ring contraction and sulfur derivatization of d5SICS, yielding the dNaM-dTPT3 UBP (Fig. 1A), results in more efficient replication in vitro (17). To explore the in vivo use of dNaM-dTPT3, we repeated the experiments with YZ3 and each of the 16 pUCX2 plasmids, but with growth in media supplemented with dNaMTP and dTPT3TP.…”

Section: Resultsmentioning

confidence: 99%

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

Zhang

Lamb

Feldman

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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153

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All natural organisms store genetic information in a four-letter, twobase-pair genetic alphabet. The expansion of the genetic alphabet with two synthetic unnatural nucleotides that selectively pair to form an unnatural base pair (UBP) would increase the information storage potential of DNA, and semisynthetic organisms (SSOs) that stably harbor this expanded alphabet would thereby have the potential to store and retrieve increased information. Toward this goal, we previously reported that Escherichia coli grown in the presence of the unnatural nucleoside triphosphates dNaMTP and d5SICSTP, and provided with the means to import them via expression of a plasmid-borne nucleoside triphosphate transporter, replicates DNA containing a single dNaM-d5SICS UBP. Although this represented an important proof-of-concept, the nascent SSO grew poorly and, more problematically, required growth under controlled conditions and even then was unable to indefinitely store the unnatural information, which is clearly a prerequisite for true semisynthetic life. Here, to fortify and vivify the nascent SSO, we engineered the transporter, used a more chemically optimized UBP, and harnessed the power of the bacterial immune response by using Cas9 to eliminate DNA that had lost the UBP. The optimized SSO grows robustly, constitutively imports the unnatural triphosphates, and is able to indefinitely retain multiple UBPs in virtually any sequence context. This SSO is thus a form of life that can stably store genetic information using a six-letter, three-base-pair alphabet.T he natural genetic alphabet is composed of four letters whose selective pairing to form two base pairs underlies the storage and retrieval of virtually all biological information. This alphabet is essentially conserved throughout nature, and has been since the last common ancestor of all life on Earth. Significant effort has been directed toward the development of an unnatural base pair (UBP), formed between two synthetic nucleotides, that functions alongside its natural counterparts (1-3), which would represent a remarkable integration of a man-made, synthetic component into one of life's most central processes. Moreover, semisynthetic organisms (SSOs) that stably harbor such a UBP in their DNA could store and potentially retrieve the increased information, and thereby lay the foundation for achieving the central goal of synthetic biology: the creation of new life forms and functions (4).For over 15 years, we have sought to develop such a UBP (1), and these efforts eventually yielded a family of predominantly hydrophobic UBPs, with that formed between dNaM and d5SICS (dNaM-d5SICS; Fig. 1A) being a particularly promising example (5-7). Despite lacking complementary hydrogen bonding, we demonstrated that the dNaM-d5SICS UBP is well replicated by a variety of DNA polymerases in vitro (7-10), and that this efficient replication is mediated by a unique mechanism that draws upon interbase hydrophobic and packing interactions (11,12). These efforts then culminated in the first pro...

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Section: Resultsmentioning

confidence: 99%

“…Triphosphates of dNaM, d5SICS, dTPT3, and dMMO2 bio were synthesized as described previously (5,7,10,17) or kindly provided by Synthorx. The dNaM-containing TK1 oligonucleotide was described previously (14).…”

Section: Methodsmentioning

confidence: 99%

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

Zhang

Lamb

Feldman

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

153

208

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“…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.…”

Section: Medicinal and Bioorganic Chemistry Of Nucleosides And Nucleomentioning

confidence: 99%

Unnatural Base Pairs for Synthetic Biology

Saito–Tarashima

Minakawa

2018

CHEMICAL & PHARMACEUTICAL BULLETIN

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“…Synthetically modified nucleosides are being used in numerous applications, such as expanding the genetic code, probing biological interactions and functions, 18 imparting favorable properties on small interfering RNAs (siRNAs), 19,20 and even creating a semi-synthetic organism with increased potential for information storage and retrieval. 21,22 Thus, ribonucleoside non-natural modifications further extend the structural repertoire and, in turn, the functional properties of RNA molecules. 23 In this scenario, a few azapurine and deazapurine analogues have been pointed out recently for several applications.…”

Section: Introductionmentioning

confidence: 99%

Structural and Energetic Impact of Non-Natural 7-Deaza-8-Azaadenine and Its 7-Substituted Derivatives on H-Bonding Potential with Uracil in RNA Molecules

et al. 2015

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Abstract:Non-natural (synthetic) nucleobases, including 7-ethynyl-and 7-triazolyl-8-aza-7-deazaadenine, have been introduced in RNA molecules for targeted applications, and have been characterized experimentally. However, no theoretical characterization of the impact of these modifications on the structure and energetics of the corresponding H-bonded base pair is available. To fill this gap, we performed quantum mechanics calculations, starting with the analysis of the impact of the 8-aza-7-deaza modification of the adenine skeleton, and we moved then to analyze the impact of the specific substituents on the modified 8-aza-7-deazaadenine. Our analysis indicates that, despite of these severe structural modifications, the H-bonding properties of the modified base pair gratifyingly replicate those of the unmodified base pair. Similar behavior is predicted when the same skeleton modifications are applied to guanine when paired to cytosine. To stress further the H-bonding pairing in the modified adenine-uracil base pair, we explored the impact of strong electron donor and electron withdrawing substituents on the C7 position. Also in this case we found minimal impact on the base pair geometry and energy, confirming the validity of this modification strategy to functionalize RNAs without perturbing its stability and biological functionality.

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Natural-like Replication of an Unnatural Base Pair for the Expansion of the Genetic Alphabet and Biotechnology Applications

Cited by 142 publications

References 32 publications

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

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

Unnatural Base Pairs for Synthetic Biology

Structural and Energetic Impact of Non-Natural 7-Deaza-8-Azaadenine and Its 7-Substituted Derivatives on H-Bonding Potential with Uracil in RNA Molecules

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