Discovery, Characterization, and Optimization of an Unnatural Base Pair for Expansion of the Genetic Alphabet

Leconte, Aaron M.; Hwang, Gil Tae; Matsuda, Shigeo; Čapek, Petr; Hari, Yoshiyuki; Romesberg, Floyd E.

doi:10.1021/ja078223d

Cited by 156 publications

(201 citation statements)

References 49 publications

Supporting

Mentioning

195

Contrasting

Unclassified

Order By: Relevance

“…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).…”

mentioning

confidence: 82%

See 1 more Smart Citation

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

150

208

View full text Add to dashboard Cite

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...

show abstract

mentioning

confidence: 82%

“…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

150

208

View full text Add to dashboard Cite

show abstract

“…Our efforts to develop predominantly hydrophobic unnatural base pairs have culminated in the identification of the pairs formed between d5SICS and either dMMO2 (28) or dNaM (11,29) (Fig. 1 shows a comparison of d5SICS-dNaM with a natural dG-dC).…”

mentioning

confidence: 99%

Efficient and sequence-independent replication of DNA containing a third base pair establishes a functional six-letter genetic alphabet

Malyshev

Dhami²,

Quach³

et al. 2012

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

Self Cite

156

130

View full text Add to dashboard Cite

The natural four-letter genetic alphabet, comprised of just two base pairs (dA-dT and dG-dC), is conserved throughout all life, and its expansion by the development of a third, unnatural base pair has emerged as a central goal of chemical and synthetic biology. We recently developed a class of candidate unnatural base pairs, exemplified by the pair formed between d5SICS and dNaM. Here, we examine the PCR amplification of DNA containing one or more d5SICS-dNaM pairs in a wide variety of sequence contexts. Under standard conditions, we show that this DNA may be amplified with high efficiency and greater than 99.9% fidelity. To more rigorously explore potential sequence effects, we used deep sequencing to characterize a library of templates containing the unnatural base pair as a function of amplification. We found that the unnatural base pair is efficiently replicated with high fidelity in virtually all sequence contexts. The results show that, for PCR and PCR-based applications, d5SICS-dNaM is functionally equivalent to a natural base pair, and when combined with dA-dT and dG-dC, it provides a fully functional six-letter genetic alphabet.expanded genetic alphabet | hydrophobic | artificial DNA | unnatural nucleotides | bioinformatics E xpansion of the genetic alphabet to include an unnatural base pair has emerged as a central goal of chemical and synthetic biology. Success would represent a remarkable integration of orthogonal synthetic components into a fundamental biological system and build the foundation for a semisynthetic organism with increased potential for information storage and retrieval (1). Moreover, the constituent unnatural nucleotides could be used to site-specifically label DNA or RNA with different functionalities of interest (2-4) and potentially revolutionize the already ubiquitous in vitro applications of nucleic acids, such as aptamer and DNA/RNAzyme selections (5, 6), PCR-based diagnostics (7, 8), and DNA-based nanomaterials and devices (9).Although many candidate unnatural base pairs have been reported (10-21), only a few are actually replicable by DNA polymerases (10,11,13,16). Moreover, it is clear that most applications will require that the unnatural base pair not only be replicated with high efficiency and fidelity but also, that replication be at least approximately independent of sequence context. Sequence dependencies would cause biased amplification and effectively preclude many uses of the unnatural base pair. No candidate unnatural base pair has been shown to be replicated without sequence bias, and thus, none can yet claim functional equivalence to a natural base pair.In general, the most promising unnatural base pair candidates currently available have been developed by pursuing one of two different strategies. The first strategy, pioneered in the work by Benner and coworkers (22), relies on the use of nucleotide analogs bearing nucleobases that pair through complementary hydrogen bonding (H-bonding) patterns that are orthogonal to those patterns of the natural pairs. Early ef...

show abstract

“…However, further replication reactions after PICS : PICS base pairing are terminated because PICS bases overlap with each other, indicating structural change in the DNA duplex. 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.…”

Section: Medicinal and Bioorganic Chemistry Of Nucleosides And Nucleomentioning

confidence: 99%

Unnatural Base Pairs for Synthetic Biology

Saito–Tarashima

Minakawa

2018

CHEMICAL & PHARMACEUTICAL BULLETIN

View full text Add to dashboard Cite

Discovery, Characterization, and Optimization of an Unnatural Base Pair for Expansion of the Genetic Alphabet

Cited by 156 publications

References 49 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

Efficient and sequence-independent replication of DNA containing a third base pair establishes a functional six-letter genetic alphabet

Unnatural Base Pairs for Synthetic Biology

Contact Info

Product

Resources

About