RESUMO
The development of unnatural base pairs (UBPs) has greatly increased the information storage capacity of DNA, allowing for transcription of unnatural RNA by the heterologously expressed T7 RNA polymerase (RNAP) in Escherichia coli. However, little is known about how UBPs are transcribed by cellular RNA polymerases. Here, we investigated how synthetic unnatural nucleotides, NaM and TPT3, are recognized by eukaryotic RNA polymerase II (Pol II) and found that Pol II is able to selectively recognize UBPs with high fidelity when dTPT3 is in the template strand and rNaMTP acts as the nucleotide substrate. Our structural analysis and molecular dynamics simulation provide structural insights into transcriptional processing of UBPs in a stepwise manner. Intriguingly, we identified a novel 3'-RNA binding site after rNaM addition, termed the swing state. These results may pave the way for future studies in the design of transcription and translation strategies in higher organisms with expanded genetic codes.
Assuntos
Eucariotos/enzimologia , RNA Polimerase II/genética , Transcrição Gênica/genética , Pareamento de Bases , Simulação de Dinâmica Molecular , RNA Polimerase II/química , RNA Polimerase II/metabolismoRESUMO
Since at least the last common ancestor of all life on Earth, genetic information has been stored in a four-letter alphabet that is propagated and retrieved by the formation of two base pairs. The central goal of synthetic biology is to create new life forms and functions, and the most general route to this goal is the creation of semi-synthetic organisms whose DNA harbours two additional letters that form a third, unnatural base pair. Previous efforts to generate such semi-synthetic organisms culminated in the creation of a strain of Escherichia coli that, by virtue of a nucleoside triphosphate transporter from Phaeodactylum tricornutum, imports the requisite unnatural triphosphates from its medium and then uses them to replicate a plasmid containing the unnatural base pair dNaM-dTPT3. Although the semi-synthetic organism stores increased information when compared to natural organisms, retrieval of the information requires in vivo transcription of the unnatural base pair into mRNA and tRNA, aminoacylation of the tRNA with a non-canonical amino acid, and efficient participation of the unnatural base pair in decoding at the ribosome. Here we report the in vivo transcription of DNA containing dNaM and dTPT3 into mRNAs with two different unnatural codons and tRNAs with cognate unnatural anticodons, and their efficient decoding at the ribosome to direct the site-specific incorporation of natural or non-canonical amino acids into superfolder green fluorescent protein. The results demonstrate that interactions other than hydrogen bonding can contribute to every step of information storage and retrieval. The resulting semi-synthetic organism both encodes and retrieves increased information and should serve as a platform for the creation of new life forms and functions.
Assuntos
Aminoácidos/química , Aminoácidos/metabolismo , Anticódon/genética , Pareamento de Bases , Escherichia coli/genética , Engenharia Genética , RNA de Transferência/genética , Biologia Sintética/métodos , Aminoácidos/genética , Diatomáceas/genética , Escherichia coli/metabolismo , Genes Reporter/genética , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Ligação de Hidrogênio , Proteínas de Transporte de Nucleosídeos/genética , Proteínas de Transporte de Nucleosídeos/metabolismo , Plasmídeos/genética , RNA Mensageiro/química , RNA Mensageiro/genética , RNA de Transferência/química , Ribossomos/metabolismoRESUMO
Natural organisms use a four-letter genetic alphabet that makes available 64 triplet codons, of which 61 are sense codons used to encode proteins with the 20 canonical amino acids. We have shown that the unnatural nucleotides dNaM and dTPT3 can pair to form an unnatural base pair (UBP) and allow for the creation of semisynthetic organisms (SSOs) with additional sense codons. Here, we report a systematic analysis of the unnatural codons. We identify nine unnatural codons that can produce unnatural protein with nearly complete incorporation of an encoded noncanonical amino acid (ncAA). We also show that at least three of the codons are orthogonal and can be simultaneously decoded in the SSO, affording the first 67-codon organism. The ability to incorporate multiple, different ncAAs site specifically into a protein should now allow the development of proteins with novel activities, and possibly even SSOs with new forms and functions.
Assuntos
Pareamento de Bases , Códon , Engenharia Genética/métodos , Nucleotídeos/química , Aminoácidos , Anticódon , Escherichia coli/genética , Proteínas de Fluorescência Verde/genética , Microrganismos Geneticamente Modificados , Nucleotídeos/genética , Proteínas Recombinantes/genéticaRESUMO
We have developed semisynthetic organisms (SSOs) that by virtue of a family of synthetic, unnatural base pairs (UBPs), store and retrieve increased information. To date, transcription in the SSOs has relied on heterologous expression of the RNA polymerase from T7 bacteriophage; here, we explore placing transcription under the control of the endogenous host multisubunit RNA polymerase. The results demonstrate that the E. coli RNA polymerase is able to transcribe DNA containing a UBP and that with the most optimal UBP identified to date it should be possible to select for increased uptake of unnatural triphosphates. These advances should facilitate the creation of next generation SSOs.
Assuntos
RNA Polimerases Dirigidas por DNA/genética , DNA/genética , Biologia Sintética , Pareamento de Bases , DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/enzimologiaRESUMO
Virtually all natural proteins are built from only 20 amino acids, and while this makes possible all the functions they perform, the ability to encode other amino acids selected for specific purposes promises to enable the discovery and production of proteins with novel functions, including therapeutic proteins with more optimal drug-like properties. The field of genetic code expansion (GCE) has for years enabled the production of such proteins for academic purposes and is now transitioning to commercialization for the production of more optimal protein therapeutics. Focusing on E. coli, we review the history and current state of the field. We also provide a review of the first generation commercialization efforts, the lessons learned, and how those lessons are guiding new efforts. With continued academic and industrial progress, GCE methodologies promise to make possible the routine optimization of proteins for therapeutic use in a way that has only previously been possible with small-molecule therapeutics.
Assuntos
Código Genético , Códon , Escherichia coli/genética , Genes BacterianosRESUMO
Through the development of unnatural base pairs that are compatible with native DNA and RNA polymerases and the ribosome, we have expanded the genetic alphabet and enabled in vitro and in vivo production of proteins containing noncanonical amino acids. However, the absence of assays to characterize transcription has prevented the deconvolution of the contributions of transcription and translation to the reduced performance of some unnatural codons. Here we show that RNA containing the unnatural nucleotides is efficiently reverse transcribed into cDNA, and we develop an assay to measure the combined fidelity of transcription and reverse transcription. With this assay, we examine the performance of a wide variety of unnatural codons, both in vitro and in the in vivo environment of a semisynthetic organism. We find that transcription is generally efficient, decoding at the ribosome is generally more challenging, and, correspondingly, sequence-dependent translation efficiency is the origin of variable codon performance.
Assuntos
RNA/metabolismo , Transcrição Reversa , Pareamento de Bases , DNA Complementar/biossíntese , RNA Polimerases Dirigidas por DNA/metabolismo , Código Genético , Ribossomos/metabolismoRESUMO
Previously, we evolved a DNA polymerase, SFM4-3, for the recognition of substrates modified at their 2' positions with a fluoro, O-methyl, or azido substituent. Here we use SFM4-3 to synthesize 2'-azido-modified DNA; we then use the azido group to attach different, large hydrophobic groups via click chemistry. We show that SFM4-3 recognizes the modified templates under standard conditions, producing natural DNA and thereby allowing amplification. To demonstrate the utility of this remarkable property, we use SFM4-3 to select aptamers with large hydrophobic 2' substituents that bind human neutrophil elastase or the blood coagulation protein factor IXa. The results indicate that SFM4-3 should facilitate the discovery of aptamers that adopt novel and perhaps more protein-like folds with hydrophobic cores that in turn allow them to access novel activities.
Assuntos
Aptâmeros de Nucleotídeos/química , DNA/química , Humanos , Interações Hidrofóbicas e HidrofílicasRESUMO
Unnatural base pairs (UBPs) have been developed and used for a variety of in vitro applications as well as for the engineering of semisynthetic organisms (SSOs) that store and retrieve increased information. However, these applications are limited by the availability of methods to rapidly and accurately determine the sequence of unnatural DNA. Here we report the development and application of the MspA nanopore to sequence DNA containing the dTPT3-dNaM UBP. Analysis of two sequence contexts reveals that DNA containing the UBP is replicated with an efficiency and fidelity similar to that of natural DNA and sufficient for use as the basis of an SSO that produces proteins with noncanonical amino acids.
Assuntos
Pareamento de Bases , Código Genético , Nanoporos , Interações Hidrofóbicas e HidrofílicasRESUMO
The arylomycins are a class of natural product antibiotics that inhibit bacterial type I signal peptidase and are under development as therapeutics. Four classes of arylomycins are known, arylomycins A-D. Previously, we reported the synthesis and analysis of representatives of the A, B, and C classes and showed that their spectrum of activity has the potential to be much broader than originally assumed. Along with a comparison of the mechanism of acquired and innate resistance, this led us to suggest that the arylomycins are latent antibiotics, antibiotics that once possessed broad-spectrum activity, but which upon examination today, have only narrow spectrum activity due to prior selection for resistance in the course of the competition with other microorganisms that drove their evolution in the first place. Interestingly, actinocarbasin, the only identified member of the arylomycin D class, has been reported to have activity against MRSA. To confirm and understand this activity, several actinocarbasin derivatives were synthesized. We demonstrate that the previously reported structure of actinocarbasin is incorrect, identify what is likely the correct scaffold, confirm that scaffold has activity against MRSA, and determine the origin of this activity.
Assuntos
Antibacterianos/análise , Antibacterianos/química , Antibacterianos/farmacologia , Testes de Sensibilidade Microbiana , Estrutura Molecular , Análise Espectral/métodos , Relação Estrutura-AtividadeRESUMO
Organisms are defined by the information encoded in their genomes, and since the origin of life this information has been encoded using a two-base-pair genetic alphabet (A-T and G-C). In vitro, the alphabet has been expanded to include several unnatural base pairs (UBPs). We have developed a class of UBPs formed between nucleotides bearing hydrophobic nucleobases, exemplified by the pair formed between d5SICS and dNaM (d5SICS-dNaM), which is efficiently PCR-amplified and transcribed in vitro, and whose unique mechanism of replication has been characterized. However, expansion of an organism's genetic alphabet presents new and unprecedented challenges: the unnatural nucleoside triphosphates must be available inside the cell; endogenous polymerases must be able to use the unnatural triphosphates to faithfully replicate DNA containing the UBP within the complex cellular milieu; and finally, the UBP must be stable in the presence of pathways that maintain the integrity of DNA. Here we show that an exogenously expressed algal nucleotide triphosphate transporter efficiently imports the triphosphates of both d5SICS and dNaM (d5SICSTP and dNaMTP) into Escherichia coli, and that the endogenous replication machinery uses them to accurately replicate a plasmid containing d5SICS-dNaM. Neither the presence of the unnatural triphosphates nor the replication of the UBP introduces a notable growth burden. Lastly, we find that the UBP is not efficiently excised by DNA repair pathways. Thus, the resulting bacterium is the first organism to propagate stably an expanded genetic alphabet.
Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Código Genético/genética , Instabilidade Genômica/genética , Nucleotídeos/genética , Nucleotídeos/metabolismo , Biologia Sintética/métodos , Proteínas de Algas/genética , Proteínas de Algas/metabolismo , Pareamento de Bases , Meios de Cultura/química , Meios de Cultura/metabolismo , Meios de Cultura/farmacologia , Reparo do DNA , Replicação do DNA , Escherichia coli/efeitos dos fármacos , Código Genético/efeitos dos fármacos , Isoquinolinas/metabolismo , Naftalenos/metabolismo , Proteínas de Transporte de Nucleotídeos/genética , Proteínas de Transporte de Nucleotídeos/metabolismo , Nucleotídeos/química , Plasmídeos/biossíntese , Plasmídeos/genética , Tionas/metabolismoRESUMO
All natural organisms store genetic information in a four-letter, two-base-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.
Assuntos
Código Genético , Sistemas CRISPR-Cas , Oligonucleotídeos , Plasmídeos , Biologia SintéticaRESUMO
Steve Benner and collaborators have recently reported an analysis of DNA containing eight nucleotide letters, the four natural letters (dG, dC, dA, and dT) and four additional letters (dP, dZ, dS, and dB). Their analysis demonstrates that the additional letters do not perturb the structure or stability of the base pairs formed between the natural letters and, remarkably, that the new base pairs, dP-dZ and dS-dB, behave virtually identically to the natural base pairs. This unprecedented result convincingly demonstrates that the thermodynamic and structural behavior previously thought to be the purview of only natural DNA is in fact not unique and can be imparted to suitably designed synthetic components. In addition, the first evidence that the eight-letter DNA can be transcribed into RNA by a mutant RNA polymerase is presented, paving the way for the transfer of more information from one biopolymer to another. Along with others working to develop unnatural DNA base pairs for both in vitro and in vivo applications, this work represents an important step toward the expansion of the genetic alphabet, a central goal of synthetic biology, and has profound implications for our understanding of the molecules and forces that can make life possible.
Assuntos
DNA , Biologia Sintética , Pareamento de Bases , RNA Polimerases Dirigidas por DNA , NucleotídeosRESUMO
For years, antibodies (Abs) have been used as a paradigm for understanding how protein structure contributes to molecular recognition. However, with the ability to evolve Abs that recognize specific chromophores, they also have great potential as models for how protein dynamics contribute to molecular recognition. We previously raised murine Abs to different chromophores and, with the use of three-pulse photon echo peak shift spectroscopy, demonstrated that the immune system is capable of producing Abs with widely varying flexibility. We now report the characterization of the complexes formed between two Abs, 5D11 and 10A6, and the chromophoric ligand that they were evolved to recognize, 8-methoxypyrene-1,3,6-trisulfonic acid (MPTS). The sequences of the Ab genes indicate that they evolved from a common precursor. We also used a variety of spectroscopic methods to probe the photophysics and dynamics of the Ab-MPTS complexes and found that they are similar to each other but distinct from previously characterized anti-MPTS Abs. Structural studies revealed that this difference likely results from a unique mode of binding in which MPTS is sandwiched between the side chain of PheH98, which interacts with the chromophore via T-stacking, and the side chain of TrpL91, which interacts with the chromophore via parallel stacking. The T-stacking interaction appears to mediate relaxation on the picosecond time scale, while the parallel stacking appears to mediate relaxation on an ultrafast, femtosecond time scale, which dominates the response. The anti-MPTS Abs thus not only demonstrate the simultaneous use of the two limiting modes of stacking for molecular recognition, but also provide a unique opportunity to characterize how dynamics might contribute to molecular recognition. Both types of stacking are common in proteins and protein complexes where they may similarly contribute to dynamics and molecular recognition.
Assuntos
Anticorpos Monoclonais/imunologia , Sítios de Ligação de Anticorpos , Pirenos/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Formação de Anticorpos , Cristalografia por Raios X , Camundongos , Modelos MolecularesRESUMO
We have created a bacterial semisynthetic organism (SSO) that retains an unnatural base pair (UBP) in its DNA, transcribes it into mRNA and tRNA with cognate unnatural codons and anticodons, and after the tRNA is charged with a noncanonical amino acid synthesizes proteins containing the noncanonical amino acid. Here, we report the first progress toward the creation of eukaryotic SSOs. After demonstrating proof-of-concept with human HEK293 cells, we show that a variety of different unnatural codon-anticodon pairs can efficiently mediate the synthesis of unnatural proteins in CHO cells. Interestingly, we find that there are both similarities and significant differences between how the prokaryotic and eukaryotic ribosomes recognize the UBP, with the eukaryotic ribosome appearing more tolerant. The results represent the first progress toward eukaryotic SSOs and, in fact, suggest that such SSOs might be able to retain more unnatural information than their bacterial counterparts.
Assuntos
Aminoácidos/genética , DNA Bacteriano/genética , Escherichia coli/genética , RNA Mensageiro/genética , RNA de Transferência/genética , Animais , Pareamento de Bases , Células CHO , Cricetulus , Código Genético , Células HEK293 , Humanos , Estrutura MolecularRESUMO
Previously, we reported the creation of a semi-synthetic organism (SSO) that stores and retrieves increased information by virtue of stably maintaining an unnatural base pair (UBP) in its DNA, transcribing the corresponding unnatural nucleotides into the codons and anticodons of mRNAs and tRNAs, and then using them to produce proteins containing noncanonical amino acids (ncAAs). Here we report a systematic extension of the effort to optimize the SSO by exploring a variety of deoxy- and ribonucleotide analogues. Importantly, this includes the first in vivo structure-activity relationship (SAR) analysis of unnatural ribonucleoside triphosphates. Similarities and differences between how DNA and RNA polymerases recognize the unnatural nucleotides were observed, and remarkably, we found that a wide variety of unnatural ribonucleotides can be efficiently transcribed into RNA and then productively and selectively paired at the ribosome to mediate the synthesis of proteins with ncAAs. The results extend previous studies, demonstrating that nucleotides bearing no significant structural or functional homology to the natural nucleotides can be efficiently and selectively paired during replication, to include each step of the entire process of information storage and retrieval. From a practical perspective, the results identify the most optimal UBP for replication and transcription, as well as the most optimal unnatural ribonucleoside triphosphates for transcription and translation. The optimized SSO is now, for the first time, able to efficiently produce proteins containing multiple, proximal ncAAs.
Assuntos
Nucleotídeos/genética , Biossíntese de Proteínas , Biologia Sintética/métodos , Transcrição Gênica , Pareamento de Bases , Desoxirribonucleotídeos/química , Desoxirribonucleotídeos/genética , Código Genético , Nucleotídeos/químicaRESUMO
At sufficient concentrations, antibiotics effectively eradicate many bacterial infections. However, during therapy, bacteria are unavoidably exposed to lower antibiotic concentrations, and sub-MIC exposure can result in a wide variety of other effects, including the induction of virulence, which can complicate therapy, or horizontal gene transfer (HGT), which can accelerate the spread of resistance genes. Bacterial type I signal peptidase (SPase) is an essential protein that acts at the final step of the general secretory pathway. This pathway is required for the secretion of many proteins, including many required for virulence, and the arylomycins are a class of natural product antibiotics that target SPase. Here, we investigated the consequences of exposing Escherichia coli cultures to sub-MIC levels of an arylomycin. Using multidimensional protein identification technology mass spectrometry, we found that arylomycin treatment inhibits the proper extracytoplasmic localization of many proteins, both those that appear to be SPase substrates and several that do not. The identified proteins are involved in a broad range of extracytoplasmic processes and include a number of virulence factors. The effects of arylomycin on several processes required for virulence were then individually examined, and we found that, at even sub-MIC levels, the arylomycins potently inhibit flagellation, motility, biofilm formation, and the dissemination of antibiotic resistance via HGT. Thus, we conclude that the arylomycins represent promising novel therapeutics with the potential to eradicate infections while simultaneously reducing virulence and the dissemination of resistance.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Bactérias/genética , Desenho de Fármacos , Resistência Microbiana a Medicamentos/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Testes de Sensibilidade Microbiana , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , VirulênciaRESUMO
The 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 and methodologies of synthetic and medical chemistry in the pursuit of synthetic biology.
Assuntos
DNA/genética , Nucleotídeos/genética , Biologia Sintética/métodos , Pareamento de Bases , Interações Hidrofóbicas e Hidrofílicas , Nucleotídeos/síntese química , Nucleotídeos/química , Relação Estrutura-AtividadeRESUMO
Vibrational spectroscopy provides a direct route to the physicochemical characterization of molecules. While both IR and Raman spectroscopy have been used for decades to provide detailed characterizations of small molecules, similar studies with proteins are largely precluded due to spectral congestion. However, the vibrational spectra of proteins do include a "transparent window", between â¼1800 and â¼2500 cm-1, and progress is now being made to develop site-specifically incorporated carbon-deuterium (C-D), cyano (CN), thiocyanate (SCN), and azide (N3) "transparent window vibrational probes" that absorb within this window and report on their environment to facilitate the characterization of proteins with small molecule-like detail. This Review opens with a brief discussion of the advantages and limitations of conventional vibrational spectroscopy and then discusses the strengths and weaknesses of the different transparent window vibrational probes, methods by which they may be site-specifically incorporated into peptides and proteins, and the physicochemical properties they may be used to study, including electrostatics, stability and folding, hydrogen bonding, protonation, solvation, dynamics, and interactions with inhibitors. The use of the probes to vibrationally image proteins and other biomolecules within cells is also discussed. We then present four case studies, focused on ketosteroid isomerase, the SH3 domain, dihydrofolate reductase, and cytochrome c, where the transparent window vibrational probes have already been used to elucidate important aspects of protein structure and function. The Review concludes by highlighting the current challenges and future potential of using transparent window vibrational probes to understand the evolution and function of proteins and other biomolecules.
Assuntos
Sondas Moleculares/química , Proteínas/química , Conformação Proteica , Espectrofotometria Infravermelho , Análise Espectral/métodos , VibraçãoRESUMO
Semisynthetic organisms (SSOs) created from Escherichia coli can replicate a plasmid containing an unnatural base pair (UBP) formed between the synthetic nucleosides dNaM and dTPT3 (dNaM-dTPT3) when the corresponding unnatural triphosphates are imported via expression of a nucleoside triphosphate transporter. The UBP can also be transcribed and used to translate proteins containing unnatural amino acids. However, UBPs are not well retained in all sequences, limiting the information that can be encoded, and are invariably lost upon extended growth. Here we explore the contributions of the E. coli DNA replication and repair machinery to the propagation of DNA containing dNaM-dTPT3 and show that replication by DNA polymerase III, supplemented with the activity of polymerase II and methyl-directed mismatch repair contribute to retention of the UBP and that recombinational repair of stalled forks is responsible for the majority of its loss. This work elucidates fundamental aspects of how bacteria replicate DNA and we use this information to reprogram the replisome of the SSO for increased UBP retention, which then allowed for the first time the construction of SSOs harboring a UBP in their chromosome.
Assuntos
Replicação do DNA , Escherichia coli/metabolismo , Pareamento de Bases , Reparo do DNA , Escherichia coli/genética , Estrutura Molecular , Plasmídeos , Biologia SintéticaRESUMO
Arylomycins are a promising class of "latent" antibacterial natural products currently in preclinical development. Access to analogues within this family has previously required a lengthy route involving multiple functional group manipulations that is costly and time-intensive on scale. This study presents a simplified route predicated on simple C-H functionalization logic that is enabled by a Cu-mediated oxidative phenol coupling that mimics the putative biosynthesis. This operationally simple macrocyclization is the largest of its kind and can be easily performed on gram scale. The application of this new route to a formal synthesis of the natural product and a collection of new analogues along with their biological evaluation is also reported.