Structural Basis of the Mispairing of an Artificially Expanded Genetic Information System

Reichenbach, Linus F.; Sobri, Ahmad Ahmad; Zaccai, Nathan R.; Agnew, Christopher; Burton, Nick; Eperon, Lucy P.; Ornellas, Sara De; Eperon, Ian C.; Brady, R.L.; Burley, Glenn A.

doi:10.1016/j.chempr.2016.11.009

Cited by 17 publications

(32 citation statements)

References 51 publications

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“…The reverse process, incorporation of dPMP opposite a templating dZ , however, was inefficient. A major drawback of the dP – dZ pair in general is the mispairing with natural nucleotides (mainly misincorporation of dGMP opposite deprotonated dZ) …”

Section: Different Ubps and Their Acceptance By Dna Polymerasesmentioning

confidence: 99%

“…Generally, the hydrogen‐bonding dP – dZ pair shows less amplification fidelity in PCR experiments compared with the hydrophobic UBPs discussed here (see values in section 2.1), mainly owing to mispairing with natural nucleotides . Therefore, in terms of orthogonality to natural pairs, it seems to be advantageous if pairing relies on a different principle.…”

Section: Comparison Of Different Ubp Candidatesmentioning

confidence: 99%

“…Generally,t he hydrogen-bonding dP-dZ pair shows less amplification fidelity in PCR experiments compared with the hydrophobic UBPs discussed here( see values in section 2.1), mainly owing to mispairing with natural nucleotides. [30,33] Therefore, in terms of orthogonality to natural pairs, it seems to be advantageous if pairingr elies on ad ifferent principle. The reported pairs that rely on hydrophobic and packing interactions rather than hydrogen bonds show low incorporation efficiencies opposite naturalp airs, leadingt oh igh fidelities in replication.…”

Section: Processing By Dna Polymerasesmentioning

confidence: 99%

“…Am ajor drawback of the dP-dZ pair in general is the mispairing with natural nucleotides (mainly misincorporation of dGMP opposite deprotonated dZ). [30,33]…”

Section: Hydrogen-bonding Ubpsmentioning

confidence: 99%

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The Structural Basis for Processing of Unnatural Base Pairs by DNA Polymerases

Marx

Betz

2020

Chemistry A European J

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Unnatural base pairs (UBPs) greatly increase the diversity of DNA and RNA, furthering their broad range of molecular biological and biotechnological approaches. Different candidates have been developed whereby alternative hydrogen‐bonding patterns and hydrophobic and packing interactions have turned out to be the most promising base‐pairing concepts to date. The key in many applications is the highly efficient and selective acceptance of artificial base pairs by DNA polymerases, which enables amplification of the modified DNA. In this Review, computational as well as experimental studies that were performed to characterize the pairing behavior of UBPs in free duplex DNA or bound to the active site of KlenTaq DNA polymerase are highlighted. The structural studies, on the one hand, elucidate how base pairs lacking hydrogen bonds are accepted by these enzymes and, on the other hand, highlight the influence of one or several consecutive UBPs on the structure of a DNA double helix. Understanding these concepts facilitates optimization of future UBPs for the manifold fields of applications.

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Section: Different Ubps and Their Acceptance By Dna Polymerasesmentioning

confidence: 99%

Section: Comparison Of Different Ubp Candidatesmentioning

confidence: 99%

Section: Processing By Dna Polymerasesmentioning

confidence: 99%

“…Am ajor drawback of the dP-dZ pair in general is the mispairing with natural nucleotides (mainly misincorporation of dGMP opposite deprotonated dZ). [30,33]…”

Section: Hydrogen-bonding Ubpsmentioning

confidence: 99%

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The Structural Basis for Processing of Unnatural Base Pairs by DNA Polymerases

Marx

Betz

2020

Chemistry A European J

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“…However, this replication is not perfect. Although mispairing between P :C or A: Z in the context of oligonucleotides results in significant decreases in melting temperature consistent with destabilization ( 25 ) and is therefore unlikely to occur during DNA replication, many natural polymerases misincorporate dGTP opposite template d Z , especially at higher pH, where deprotonation of d Z gives an anionic nucleobase that forms a complementary hydrogen bond to G ( 26 , 27 ). Further, incorporation stops after three or four consecutive P:Z pairs are incorporated ( 28 ).…”

Section: Introductionmentioning

confidence: 99%

Snapshots of an evolved DNA polymerase pre- and post-incorporation of an unnatural nucleotide

Singh

Laos

Hoshika

et al. 2018

Nucleic Acids Research

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The next challenge in synthetic biology is to be able to replicate synthetic nucleic acid sequences efficiently. The synthetic pair, 2-amino-8-(1-beta-d-2′- deoxyribofuranosyl) imidazo [1,2-a]-1,3,5-triazin-[8H]-4-one (trivially designated P) with 6-amino-3-(2′-deoxyribofuranosyl)-5-nitro-1H-pyridin-2-one (trivially designated Z), is replicated by certain Family A polymerases, albeit with lower efficiency. Through directed evolution, we identified a variant KlenTaq polymerase (M444V, P527A, D551E, E832V) that incorporates dZTP opposite P more efficiently than the wild-type enzyme. Here, we report two crystal structures of this variant KlenTaq, a post-incorporation complex that includes a template-primer with P:Z trapped in the active site (binary complex) and a pre-incorporation complex with dZTP paired to template P in the active site (ternary complex). In forming the ternary complex, the fingers domain exhibits a larger closure angle than in natural complexes but engages the template-primer and incoming dNTP through similar interactions. In the binary complex, although many of the interactions found in the natural complexes are retained, there is increased relative motion of the thumb domain. Collectively, our analyses suggest that it is the post-incorporation complex for unnatural substrates that presents a challenge to the natural enzyme and that more efficient replication of P:Z pairs requires a more flexible polymerase.

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Building better enzymes: Molecular basis of improved non‐natural nucleobase incorporation by an evolved DNA polymerase

et al. 2019

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Obtaining semisynthetic microorganisms that exploit the information density of “hachimoji” DNA requires access to engineered DNA polymerases. A KlenTaq variant has been reported that incorporates the “hachimoji” P:Z nucleobase pair with a similar efficiency to that seen for Watson–Crick nucleobase incorporation by the wild type (WT) KlenTaq DNA polymerase. The variant polymerase differs from WT KlenTaq by only four amino acid substitutions, none of which are located within the active site. We now report molecular dynamics (MD) simulations on a series of binary complexes aimed at elucidating the contributions of the four amino acid substitutions to altered catalytic activity. These simulations suggest that WT KlenTaq is insufficiently flexible to be able to bind AEGIS DNA correctly, leading to the loss of key protein/DNA interactions needed to position the binary complex for efficient incorporation of the “hachimoji” Z nucleobase. In addition, we test literature hypotheses about the functional roles of each amino acid substitution and provide a molecular description of how individual residue changes contribute to the improved activity of the KlenTaq variant. We demonstrate that MD simulations have a clear role to play in systematically screening DNA polymerase variants capable of incorporating different types of nonnatural nucleobases thereby limiting the number that need to be characterized by experiment. It is now possible to build DNA molecules containing nonnatural nucleobase pairs in addition to A:T and G:C. Exploiting this development in synthetic biology requires engineered DNA polymerases that can replicate nonnatural nucleobase pairs. Computational studies on a DNA polymerase variant reveal how amino acid substitutions outside of the active site yield an enzyme that replicates nonnatural nucleobase pairs with high efficiency. This work will facilitate efforts to obtain bacteria possessing an expanded genetic alphabet.

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Structural Basis of the Mispairing of an Artificially Expanded Genetic Information System

Cited by 17 publications

References 51 publications

The Structural Basis for Processing of Unnatural Base Pairs by DNA Polymerases

The Structural Basis for Processing of Unnatural Base Pairs by DNA Polymerases

Snapshots of an evolved DNA polymerase pre- and post-incorporation of an unnatural nucleotide

Building better enzymes: Molecular basis of improved non‐natural nucleobase incorporation by an evolved DNA polymerase

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