Title: A unified Watson-Crick geometry drives transcription of six-letter expanded DNA alphabets by E. coli RNA polymerase
Abstract
Artificially Expanded Genetic Information Systems (AEGIS) add independently replicable unnatural nucleotide pairs to the natural G:C and A:T/U pairs found in native DNA, joining the unnatural pairs through alternative modes of hydrogen bonding. Whether and how AEGIS pairs are recognized and processed by multi-subunit cellular RNA polymerases (RNAPs) remains unknown. Here, we show thatE. coliRNAP selectively recognizes unnatural nucleobases in a six-letter expanded genetic system. High-resolution cryo-EM structures of three RNAP elongation complexes containing template-substrate UBPs reveal the shared principles behind the recognition of AEGIS and natural base pairs. In these structures, RNAPs are captured in an active state, poised to perform the chemistry step. At this point, the unnatural base pair adopts a Watson-Crick geometry, and the trigger loop is folded into an active conformation, indicating that the mechanistic principles underlying recognition and incorporation of natural base pairs also apply to AEGIS unnatural base pairs. These data validate the design philosophy of AEGIS unnatural basepairs. Further, we provide structural evidence supporting a long-standing hypothesis that pair mismatch during transcription occurs via tautomerization. Together, our work highlights the importance of Watson-Crick complementarity underlying the design principles of AEGIS base pair recognition.
Shukla, Madhura S.; Hoshika, Shuichi; Benner, Steven A.; Georgiadis, Millie M.(
, Philosophical Transactions of the Royal Society B: Biological Sciences)
The first structural model of duplex DNA reported in 1953 by Watson & Crick presented the double helix in B-form, the form that genomic DNA exists in much of the time. Thus, artificial DNA seeking to mimic the properties of natural DNA should also be able to adopt B-form. Using a host–guest system in which Moloney murine leukemia virus reverse transcriptase serves as the host and DNA as the guests, we determined high-resolution crystal structures of three complexes including 5′-CTTBPPBBSSZZSAAG, 5′-CTTSSPBZPSZBBAAG and 5′-CTTZZPBSBSZPPAAG with 10 consecutive unnatural nucleobase pairs in B-form within self-complementary 16 bp duplex oligonucleotides. We refer to this ALternative Isoinformational ENgineered (ALIEN) genetic system containing two nucleobase pairs (P:Z, pairing 2-amino-imidazo-[1,2-a]-1,3,5-triazin-(8H)-4-one with 6-amino-5-nitro-(1H)-pyridin-2-one, andB:S, 6-amino-4-hydroxy-5-(1H)-purin-2-one with 3-methyl-6-amino-pyrimidin-2-one) as ALIEN DNA. We characterized both position- and sequence-specific helical, nucleobase pair and dinucleotide step parameters ofP:ZandB:Spairs in the context of B-form DNA. We conclude that ALIEN DNA exhibits structural features that vary with sequence. Further,Zcan participate in alternative stacking modes within a similar sequence context as captured in two different structures. This finding suggests that ALIEN DNA may have a larger repertoire of B-form structures than natural DNA.
This article is part of the theme issue ‘Reactivity and mechanism in chemical and synthetic biology’.
In this work hydrogen bonding in a diverse set of 36 unnatural and the three natural Watson Crick base pairs adenine (A)–thymine (T), adenine (A)–uracil (U) and guanine (G)–cytosine (C) was assessed utilizing local vibrational force constants derived from the local mode analysis, originally introduced by Konkoli and Cremer as a unique bond strength measure based on vibrational spectroscopy. The local mode analysis was complemented by the topological analysis of the electronic density and the natural bond orbital analysis. The most interesting findings of our study are that (i) hydrogen bonding in Watson Crick base pairs is not exceptionally strong and (ii) the N–H⋯N is the most favorable hydrogen bond in both unnatural and natural base pairs while O–H⋯N/O bonds are the less favorable in unnatural base pairs and not found at all in natural base pairs. In addition, the important role of non-classical C–H⋯N/O bonds for the stabilization of base pairs was revealed, especially the role of C–H⋯O bonds in Watson Crick base pairs. Hydrogen bonding in Watson Crick base pairs modeled in the DNA via a QM/MM approach showed that the DNA environment increases the strength of the central N–H⋯N bond and the C–H⋯O bonds, and at the same time decreases the strength of the N–H⋯O bond. However, the general trends observed in the gas phase calculations remain unchanged. The new methodology presented and tested in this work provides the bioengineering community with an efficient design tool to assess and predict the type and strength of hydrogen bonding in artificial base pairs.
Xeno-nucleic acids (XNAs) are synthetic genetic polymers with backbone structures composed of non-ribose or non-deoxyribose sugars. Phosphonomethylthreosyl nucleic acid (pTNA), a type of XNA that does not base pair with DNA or RNA, has been suggested as a possible genetic material for storing synthetic biology information in cells. A critical step in this process is the synthesis of XNA episomes using laboratory-evolved polymerases to copy DNA information into XNA. Here, we investigate the polymerase recognition of pTNA nucleotides using X-ray crystallography to capture the post-catalytic complex of engineered polymerases following the sequential addition of two pTNA nucleotides onto the 3′-end of a DNA primer. High-resolution crystal structures reveal that the polymerase mediates Watson–Crick base pairing between the extended pTNA adducts and the DNA template. Comparative analysis studies demonstrate that the sugar conformation and backbone position of pTNA are structurally more similar to threose nucleic acid than DNA even though pTNA and DNA share the same six-atom backbone repeat length. Collectively, these findings provide new insight into the structural determinants that guide the enzymatic synthesis of an orthogonal genetic polymer, and may lead to the discovery of new variants that function with enhanced activity.
Moe, May Myat; Benny, Jonathan; Liu, Jianbo(
, Physical Chemistry Chemical Physics)
It has been shown previously in protonated, deprotonated and ionized guanine–cytosine base pairs that intra-base pair proton transfer from the N1–H at the Watson–Crick edge of guanine to the complementary nucleobase prompts non-statistical dissociation of the base-pair system, and the dissociation of a proton-transferred base-pair structure is kinetically more favored than that of the starting, conventional base-pair structure. However, the fundamental chemistry underlying this anomalous and intriguing kinetics has not been completely revealed, which warrants the examination of more base-pair systems in different structural contexts in order to derive a generalized base-pair structure–kinetics correlation. The purpose of the present work is to expand the investigation to the non-canonical homodimeric and heterodimeric radical cations of 9-methylguanine (9MG) and 9-methyl-8-oxoguanine (9MOG), i.e. , [9MG·9MG]˙ + , [9MOG·9MG]˙ + and [9MOG·9MOG]˙ + . Experimentally, collision-induced dissociation tandem mass spectrometry coupled with an electrospray ionization (ESI) source was used for the formation of base-pair radical cations, followed by detection of dissociation product ions and cross sections in the collisions with Xe gas under single ion–molecule collision conditions and as a function of the center-of-mass collision energy. Computationally, density functional theory and coupled cluster theory were used to calculate and identify probable base-pair structures and intra-base pair proton transfer and hydrogen transfer reactions, followed by kinetics modeling to explore the properties of dissociation transition states and kinetic factors. The significance of this work is twofold: it provides insight into base-pair opening kinetics in three biologically-important, non-canonical systems upon oxidative and ionization damage; and it links non-statistical dissociation to intra-base pair proton-transfer originating from the N1–H at the Watson–Crick edge of 8-oxoguanine, enhancing understanding towards the base-pair fragmentation assisted by proton transfer.
Mei, Hui; Wang, Yajun; Yik, Eric J.; Chaput, John C.(
, Biopolymers)
Abstract
Synthetic genetics is an area of synthetic biology that aims to extend the properties of heredity and evolution to artificial genetic polymers, commonly known as xeno‐nucleic acids or XNAs. In addition to establishing polymerases that are able to convert genetic information back and forth between DNA and XNA, efforts are underway to construct XNAs with expanded chemical functionality. α‐L‐Threose nucleic acid (TNA), a type of XNA that is recalcitrant to nuclease digestion and amenable to Darwinian evolution, provides a model system for developing XNAs with functional groups that are not present in natural DNA and RNA. Here, we describe the synthesis and polymerase activity of a cytidine TNA triphosphate analog (6‐phenyl‐pyrrolocytosine, tCpTP) that maintains Watson‐Crick base pairing with guanine. Polymerase‐mediated primer extension assays show that tCpTP is an efficient substrate for Kod‐RI, a DNA‐dependent TNA polymerase developed to explore the functional properties of TNA byin vitroselection. Fidelity studies reveal that a cycle of TNA synthesis and reverse transcription occurs with 99.9% overall fidelity when tCpTP and 7‐deaza‐tGTP are present as TNA substrates. This result expands the toolkit of TNA building blocks available forin vitroselection.
Oh, Juntaek, Shan, Zelin, Hoshika, Shuichi, Xu, Jun, Chong, Jenny, Benner, Steven A., Lyumkis, Dmitry, and Wang, Dong. A unified Watson-Crick geometry drives transcription of six-letter expanded DNA alphabets by E. coli RNA polymerase. Nature Communications 14.1 Web. doi:10.1038/s41467-023-43735-9.
Oh, Juntaek, Shan, Zelin, Hoshika, Shuichi, Xu, Jun, Chong, Jenny, Benner, Steven A., Lyumkis, Dmitry, and Wang, Dong.
"A unified Watson-Crick geometry drives transcription of six-letter expanded DNA alphabets by E. coli RNA polymerase". Nature Communications 14 (1). Country unknown/Code not available: Nature Publishing Group. https://doi.org/10.1038/s41467-023-43735-9.https://par.nsf.gov/biblio/10479230.
@article{osti_10479230,
place = {Country unknown/Code not available},
title = {A unified Watson-Crick geometry drives transcription of six-letter expanded DNA alphabets by E. coli RNA polymerase},
url = {https://par.nsf.gov/biblio/10479230},
DOI = {10.1038/s41467-023-43735-9},
abstractNote = {Abstract Artificially Expanded Genetic Information Systems (AEGIS) add independently replicable unnatural nucleotide pairs to the natural G:C and A:T/U pairs found in native DNA, joining the unnatural pairs through alternative modes of hydrogen bonding. Whether and how AEGIS pairs are recognized and processed by multi-subunit cellular RNA polymerases (RNAPs) remains unknown. Here, we show thatE. coliRNAP selectively recognizes unnatural nucleobases in a six-letter expanded genetic system. High-resolution cryo-EM structures of three RNAP elongation complexes containing template-substrate UBPs reveal the shared principles behind the recognition of AEGIS and natural base pairs. In these structures, RNAPs are captured in an active state, poised to perform the chemistry step. At this point, the unnatural base pair adopts a Watson-Crick geometry, and the trigger loop is folded into an active conformation, indicating that the mechanistic principles underlying recognition and incorporation of natural base pairs also apply to AEGIS unnatural base pairs. These data validate the design philosophy of AEGIS unnatural basepairs. Further, we provide structural evidence supporting a long-standing hypothesis that pair mismatch during transcription occurs via tautomerization. Together, our work highlights the importance of Watson-Crick complementarity underlying the design principles of AEGIS base pair recognition.},
journal = {Nature Communications},
volume = {14},
number = {1},
publisher = {Nature Publishing Group},
author = {Oh, Juntaek and Shan, Zelin and Hoshika, Shuichi and Xu, Jun and Chong, Jenny and Benner, Steven A. and Lyumkis, Dmitry and Wang, Dong},
}
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