The Expanded Genetic Alphabet

Malyshev, Denis A.; Romesberg, Floyd E.

doi:10.1002/anie.201502890

Cited by 184 publications

(186 citation statements)

References 209 publications

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“…6 In contrast to the natural nucleotides, the pairing of these unnatural nucleotides relies on hydrophobic and packing forces as opposed to complementary hydrogen bonding. While the development of this UBP required years of optimization, 8 the completely unnatural mode of pairing between the nucleobases of the UBP is expected to bestow them with an inherent orthogonality, with respect to their mispairing with the natural nucleotides and to their recognition by proteins that recognize the natural triphosphates (including the many proteins that interact with the triphosphates outside of information storage and retrieval). In 2014, with the aid of a nucleoside triphosphate transporter to import the unnatural triphosphates, we showed that a precursor of the d NaM -d TPT3 UBP may be retained within the DNA of growing and dividing E. coli , 12 and more recently, with the use of SpCas9 directed to eliminate sequences that have lost the UBP, we showed that d NaM -d TPT3 is retained in the DNA of the SSO with fidelity approaching that of a natural base pair.…”

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confidence: 99%

Synthetic Biology Parts for the Storage of Increased Genetic Information in Cells

2017

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To bestow cells with novel forms and functions, the goal of synthetic biology, we have developed the unnatural nucleoside triphosphates dNaMTP and dTPT3TP, which form an unnatural base pair (UBP) and expand the genetic alphabet. While the UBP may be retained in the DNA of a living cell, its retention is sequence-dependent. We now report a steady-state kinetic characterization of the rate with which the Klenow fragment of E. coli DNA polymerase I synthesizes the UBP and its mispairs in a variety of sequence contexts. Correct UBP synthesis is as efficient as for a natural base pair, except in one sequence context, and in vitro performance is correlated with in vivo performance. The data elucidate the determinants of efficient UBP synthesis, show that the dNaM-dTPT3 UBP is the first generally recognized as a natural base pair, and importantly, demonstrate that dNaMTP and dTPT3TP are well optimized and standardized parts for the expansion of the genetic alphabet.

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confidence: 99%

Synthetic Biology Parts for the Storage of Increased Genetic Information in Cells

2017

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“…This intercalation appears to be driven by favorable stacking of the large aromatic surfaces of each d PICS nucleobase, and has been observed with other nucleotides bearing nucleobase analogs with extended aromatic surface area. 18 We hypothesized that the same mode of pairing occurs at the primer terminus, which we envisioned would account for the efficient rates of UBP synthesis, but also the inefficient rates of continued extension, as de-intercalation would be required to appropriately position the primer terminus.…”

Section: A Structural Interludementioning

confidence: 99%

Expansion of the Genetic Alphabet: A Chemist’s Approach to Synthetic Biology

Feldman

Romesberg

2017

Acc. Chem. Res.

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ConspectusThe information available to any organism is encoded in a four nucleotide, two base pair genetic code. Since its earliest days, the field of synthetic biology has endeavored to impart organisms with novel attributes and functions, and perhaps the most fundamental approach to this goal is the creation of a fifth and sixth nucleotide that pair to form a third, unnatural base pair (UBP) and thus allow for the storage and retrieval of increased information. Achieving this goal, by definition, requires synthetic chemistry to create unnatural nucleotides and a medicinal chemistry-like approach to guide their optimization. With this perspective, almost 20 years ago we began designing unnatural nucleotides with the ultimate goal of developing UBPs that function in vivo, and thus serve as the foundation of semi-synthetic organisms (SSOs) capable of storing and retrieving increased information. From the beginning, our efforts focused on the development of nucleotides that bear predominantly hydrophobic nucleobases and thus that pair not based on the complementary hydrogen bonds that are so prominent among the natural base pairs but rather via hydrophobic and packing interactions. It was envisioned that such a pairing mechanism would provide a basal level of selectivity against pairing with natural nucleotides, which we expected would be the greatest challenge; however, this choice mandated starting with analogs that have little or no homology to their natural counterparts and that, perhaps not surprisingly, performed poorly. Progress toward their optimization was driven by the construction of structure–activity relationships, initially from in vitro steady-state kinetic analysis, then later from pre-steady-state and PCR-based assays, and ultimately from performance in vivo, with the results augmented three times with screens that explored combinations of the unnatural nucleotides that were too numerous to fully characterize individually. The structure–activity relationship data identified multiple features required by the UBP, and perhaps most prominent among them was a substituent ortho to the glycosidic linkage that is capable of both hydrophobic packing and hydrogen bonding, and nucleobases that stably stack with flanking natural nucleobases in lieu of the potentially more stabilizing stacking interactions afforded by cross strand intercalation. Most importantly, after the examination of hundreds of unnatural nucleotides and thousands of candidate UBPs, the efforts ultimately resulted in the identification of a family of UBPs that are well recognized by DNA polymerases when incorporated into DNA and that have been used to create SSOs that store and retrieve increased information. In addition to achieving a longstanding goal of synthetic biology, the results have important implications for our understanding of both the molecules and forces that can underlie biological processes, so long considered the purview of molecules benefiting from eons of evolution, and highlight the promise of applying the approaches an...

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“…While the idea to create a third base pair to enhance the genetic alphabet dates back to 1962 [41], it took numerous iterations of additional base pairs to attain some that were able to be incorporated into normal DNA strands and amplifiable with satisfactory fidelity without the risk of mispairing with the natural nucleobases [42]. By now, three different groups published well-characterized novel base pairs with properties that allow their use in aptamer selections: Romesberg's group is working with dNaMd5SICS (dNaM: 1,4-Anhydro-2-deoxy-1-C-(3-methoxy-2-naphthalenyl)-(1R)-D-erythro-pentitol, d5SICS: 2-((2R,4R,5R)-tetrahydro-4-hydroxy-5-(hydroxymethyl) furan-2-yl)-6-methylisoquinoline-1(2H)-thione), Hirao and co-workers have published dDs-dPx (Ds: 7-(2-thienyl)imidazo [4,5-b]pyridine, Px: 2-nitro-4-propynylpyrrole), and Benner's laboratory is utilizing the dZ-dP (dZ: 6-amino-5-nitro-3- (Figure 4).…”

Section: Aptamers With An Extended Genetic Alphabetmentioning

confidence: 99%

“…(c) The dDs-dPx base pair from Hirao's group, of which dDs has been used for aptamer selections [43 ]. (d) The d5SICS-dNaM base pair from Romesberg's group [42].…”

Section: Aptamers With An Extended Genetic Alphabetmentioning

confidence: 99%