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1.
ACS Synth Biol ; 11(11): 3874-3885, 2022 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-36278399

RESUMO

Threose nucleic acid (TNA) is considered a potential RNA progenitor due to its chemical simplicity, base pairing property, and capability of folding into a functional tertiary structure. However, it is unknown whether the functional property can be maintained during transition from TNA to RNA. Here, we use a toggle in vitro selection to identify nucleic acid catalyst sequences that are active in both TNA and RNA backbones. One such nucleic acid enzyme with exchangeable backbone (CAMELEON) catalyzes an RNA cleavage reaction when prepared as TNA (T) and RNA (R). Further biochemical characterization reveals that CAMELEON R and T exhibit different catalytic behaviors such as rate enhancement and magnesium dependence. Structural probing and mutagenesis experiments suggest that they likely fold into distinct tertiary structures. This work demonstrates that the catalytic activity can be preserved during backbone transition from TNA to RNA and provides further experimental support for TNA as an RNA precursor in evolution.


Assuntos
Ácidos Nucleicos , RNA Catalítico , Ácidos Nucleicos/química , RNA/genética , RNA/química , Tetroses/química , Pareamento de Bases , Conformação de Ácido Nucleico , RNA Catalítico/genética
2.
Nat Chem ; 14(3): 350-359, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34916596

RESUMO

Threose nucleic acid has been considered a potential evolutionary progenitor of RNA because of its chemical simplicity, base pairing properties and capacity for higher-order functions such as folding and specific ligand binding. Here we report the in vitro selection of RNA-cleaving threose nucleic acid enzymes. One such enzyme, Tz1, catalyses a site-specific RNA-cleavage reaction with an observed pseudo first-order rate constant (kobs) of 0.016 min-1. The catalytic activity of Tz1 is maximal at 8 mM Mg2+ and remains relatively constant from pH 5.3 to 9.0. Tz1 preferentially cleaves a mutant epidermal growth factor receptor RNA substrate with a single point substitution, while leaving the wild-type intact. We demonstrate that Tz1 mediates selective gene silencing of the mutant epidermal growth factor receptor in eukaryotic cells. The identification of catalytic threose nucleic acids provides further experimental support for threose nucleic acid as an ancestral genetic and functional material. The demonstration of Tz1 mediating selective knockdown of intracellular RNA suggests that functional threose nucleic acids could be developed for future biomedical applications.


Assuntos
Ácidos Nucleicos , Receptores ErbB/metabolismo , Conformação de Ácido Nucleico , Ácidos Nucleicos/química , Mutação Puntual , RNA/química , Tetroses
3.
ACS Synth Biol ; 10(11): 3190-3199, 2021 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-34739228

RESUMO

Synthetic genetic polymers (xeno-nucleic acids, XNAs) have the potential to transition aptamers from laboratory tools to therapeutic agents, but additional functionality is needed to compete with antibodies. Here, we describe the evolution of a biologically stable artificial genetic system composed of α-l-threofuranosyl nucleic acid (TNA) that facilitates the production of backbone- and base-modified aptamers termed "threomers" that function as high quality protein capture reagents. Threomers were discovered against two prototypical protein targets implicated in human diseases through a combination of in vitro selection and next-generation sequencing using uracil nucleotides that are uniformly equipped with aromatic side chains commonly found in the paratope of antibody-antigen crystal structures. Kinetic measurements reveal that the side chain modifications are critical for generating threomers with slow off-rate binding kinetics. These findings expand the chemical space of evolvable non-natural genetic systems to include functional groups that enhance protein target binding by mimicking the structural properties of traditional antibodies.


Assuntos
Aptâmeros de Nucleotídeos/química , Ácidos Nucleicos/química , Polímeros/química , Tetroses/química , Anticorpos/química , Cinética , Proteínas/química
4.
J Microbiol Biotechnol ; 31(10): 1446-1454, 2021 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-34409950

RESUMO

Herein, we cloned and expressed an endo-ß-1,4-glucanase gene (celA1805) from Bacillus subtilis B111 in Escherichia coli. The recombinant celA1805 contains a glycosyl hydrolase (GH) family 8 domain and shared 76.8% identity with endo-1,4-ß-glucanase from Bacillus sp. KSM-330. Results showed that the optimal pH and temperature of celA1805 were 6.0 and 50°C, respectively, and it was stable at pH 3-9 and temperature ≤50°C. Metal ions slightly affected enzyme activity, but chemical agents generally inhibited enzyme activity. Moreover, celA1805 showed a wide substrate specificity to CMC, barley ß-glucan, lichenin, chitosan, PASC and avicel. The Km and Vmax values of celA1805 were 1.78 mg/ml and 50.09 µmol/min/mg. When incubated with cellooligosaccharides ranging from cellotriose to cellopentose, celA1805 mainly hydrolyzed cellotetrose (G4) and cellopentose (G5) to cellose (G2) and cellotriose (G3), but hardly hydrolyzed cellotriose. The concentrations of reducing sugars saccharified by celA1805 from wheat straw, rape straw, rice straw, peanut straw, and corn straw were increased by 0.21, 0.51, 0.26, 0.36, and 0.66 mg/ml, respectively. The results obtained in this study suggest potential applications of celA1805 in biomass saccharification.


Assuntos
Bacillus subtilis/enzimologia , Celulase/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Biomassa , Celulose/análogos & derivados , Clonagem Molecular , Estabilidade Enzimática , Glucanos , Hidrólise , Caules de Planta , Estrutura Terciária de Proteína , Proteínas Recombinantes , Microbiologia do Solo , Especificidade por Substrato , Tetroses
5.
J Am Chem Soc ; 143(21): 8154-8163, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34028252

RESUMO

Threose nucleic acid (TNA) has been considered a potential RNA progenitor in evolution due to its chemical simplicity and base pairing property. Catalytic TNA sequences with RNA ligase activities might have facilitated the transition to the RNA world. Here we report the isolation of RNA ligase TNA enzymes by in vitro selection. The identified TNA enzyme T8-6 catalyzes the formation of a 2'-5' phosphoester bond between a 2',3'-diol and a 5'-triphosphate group, with a kobs of 1.1 × 10-2 min-1 (40 mM Mg2+, pH 9.0). For efficient reaction, T8-6 requires UA|GA at the ligation junction and tolerates variations at other substrate positions. Functional RNAs such as hammerhead ribozyme can be prepared by T8-6-catalyzed ligation, with site-specific introduction of a 2'-5' linkage. Together, this work provides experimental support for TNA as a plausible pre-RNA genetic polymer and also offers an alternative molecular tool for biotechnology.


Assuntos
Ácidos Nucleicos/metabolismo , RNA Ligase (ATP)/metabolismo , Tetroses/metabolismo , Conformação de Ácido Nucleico , Ácidos Nucleicos/química , RNA Ligase (ATP)/química , Tetroses/química
6.
Angew Chem Int Ed Engl ; 60(19): 10526-10530, 2021 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-33644959

RESUMO

The structure of life's first genetic polymer is a question of intense ongoing debate. The "RNA world theory" suggests RNA was life's first nucleic acid. However, ribonucleotides are complex chemical structures, and simpler nucleic acids, such as threose nucleic acid (TNA), can carry genetic information. In principle, nucleic acids like TNA could have played a vital role in the origins of life. The advent of any genetic polymer in life requires synthesis of its monomers. Here we demonstrate a high-yielding, stereo-, regio- and furanosyl-selective prebiotic synthesis of threo-cytidine 3, an essential component of TNA. Our synthesis uses key intermediates and reactions previously exploited in the prebiotic synthesis of the canonical pyrimidine ribonucleoside cytidine 1. Furthermore, we demonstrate that erythro-specific 2',3'-cyclic phosphate synthesis provides a mechanism to photochemically select TNA cytidine. These results suggest that TNA may have coexisted with RNA during the emergence of life.


Assuntos
Citidina/síntese química , Ácidos Nucleicos/síntese química , Tetroses/síntese química , Configuração de Carboidratos , Citidina/química , Ácidos Nucleicos/química , Processos Fotoquímicos , Tetroses/química
7.
J Phys Chem Lett ; 12(4): 1352-1359, 2021 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-33507076

RESUMO

Rotational spectroscopy provides the most powerful means of identifying molecules of biological interest in the interstellar medium (ISM), but despite their importance, the detection of carbohydrates has remained rather elusive. Here, we present a comprehensive Fourier transform rotational spectroscopic study of elusive erythrulose, a sugar building block likely to be present in the ISM, employing a novel method of transferring the hygroscopic oily carbohydrate into the gas phase. The high sensitivity of the experiment allowed the rotational spectra of all monosubstituted isotopologue species of 13C-12C3H8O4 to be recorded, which, together with quantum chemical calculations, enabled us to determine their equilibrium geometries (reSE) with great precision. Searches employing the new experimental data for erythrulose have been undertaken in different ISM regions, so far including the cold areas Barnard 1, the pre-stellar core TMC-1, Sagittarius B2. Although no lines of erythrulose were found, this data will serve to enable future searches and possible detections in other ISM regions.


Assuntos
Meio Ambiente Extraterreno/química , Tetroses/química , Fenômenos Astronômicos , Teoria da Densidade Funcional , Espectroscopia de Infravermelho com Transformada de Fourier
8.
Int J Biol Macromol ; 171: 166-176, 2021 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-33421464

RESUMO

Exploring new multifunctional enzymes and understanding the mechanisms of catalytic promiscuity will be of enormous industrial and academic values. In the present study, we reported the discovery and characterization of a multifunctional enzyme BSGH13 from Bacillus subtilis BS-5. Remarkably, BSGH13 possessed α-amylase, endoglucanase, and xylanase activities. To our knowledge, this was the first report on an amylase from Bacillus species having additional endoglucanase and xylanase activities. Subsequently, we analyzed the effects of aromatic residues substitution at each site of the active site architecture on ligand-binding affinity and catalytic specificity of BSGH13 by a combination of virtual mutation and site-directed mutagenesis approaches. Our results indicated that the introduction of aromatic amino acids Phe or Trp at the positions L182 and L183 altered the local interaction network of BSGH13 towards different substrates, thus changing the multifunctional properties of BSGH13. Moreover, we provided an expanded perspective on studies of multifunctional enzymes.


Assuntos
Bacillus subtilis/química , Proteínas de Bactérias/química , Celulase/química , Endo-1,4-beta-Xilanases/química , alfa-Amilases/química , Substituição de Aminoácidos , Bacillus subtilis/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Celulase/genética , Celulase/metabolismo , Celulose/análogos & derivados , Celulose/química , Celulose/metabolismo , Clonagem Molecular , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Maltose/análogos & derivados , Maltose/química , Maltose/metabolismo , Modelos Moleculares , Mutação , Fenilalanina/química , Fenilalanina/genética , Fenilalanina/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia Estrutural de Proteína , Relação Estrutura-Atividade , Especificidade por Substrato , Tetroses/química , Tetroses/metabolismo , Triptofano/química , Triptofano/genética , Triptofano/metabolismo , Xilanos/química , Xilanos/metabolismo , alfa-Amilases/genética , alfa-Amilases/metabolismo
9.
Biopolymers ; 112(1): e23388, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32615644

RESUMO

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, tCp TP) that maintains Watson-Crick base pairing with guanine. Polymerase-mediated primer extension assays show that tCp TP is an efficient substrate for Kod-RI, a DNA-dependent TNA polymerase developed to explore the functional properties of TNA by in vitro selection. Fidelity studies reveal that a cycle of TNA synthesis and reverse transcription occurs with 99.9% overall fidelity when tCp TP and 7-deaza-tGTP are present as TNA substrates. This result expands the toolkit of TNA building blocks available for in vitro selection.


Assuntos
Citidina/química , DNA Polimerase Dirigida por DNA/metabolismo , Ácidos Nucleicos/metabolismo , Pareamento de Bases , Ácidos Nucleicos/química , Polifosfatos/química , Biologia Sintética/métodos , Tetroses/química
10.
J Am Chem Soc ; 143(42): 17761-17768, 2021 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-34637287

RESUMO

Expanding the chemical space of evolvable non-natural genetic polymers (XNAs) to include functional groups that enhance protein target binding affinity offers a promising route to therapeutic aptamers with high biological stability. Here we describe the chemical synthesis and polymerase recognition of 10 chemically diverse functional groups introduced at the C-5 position of α-l-threofuranosyl uridine nucleoside triphosphate (tUTP). We show that the set of tUTP substrates is universally recognized by the laboratory-evolved polymerase Kod-RSGA. Insights into the mechanism of TNA synthesis were obtained from a high-resolution X-ray crystal structure of the postcatalytic complex bound to the primer-template duplex. A structural analysis reveals a large cavity in the enzyme active site that can accommodate the side chain of C-5-modified tUTP substrates. Our findings expand the chemical space of evolvable nucleic acid systems by providing a synthetic route to artificial genetic polymers that are uniformly modified with diversity-enhancing functional groups.


Assuntos
DNA Polimerase Dirigida por DNA/química , Tetroses/química , Uridina Trifosfato/análogos & derivados , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Cristalografia por Raios X , DNA Polimerase Dirigida por DNA/metabolismo , Nucleosídeos/química , Ligação Proteica , Tetroses/síntese química , Tetroses/metabolismo , Thermococcus/enzimologia , Uridina Trifosfato/síntese química , Uridina Trifosfato/metabolismo
11.
Artigo em Inglês | MEDLINE | ID: mdl-33063584

RESUMO

The lack of effective methods to perform direct ß-selective glycosylation reactions with 2-deoxy-1,4-dithio-D-erythro-pentofuranosides has long been a significant stumbling block for the multi-gram synthesis of 4'-thio-2'-deoxy nucleosides. In addition, previously reported methods for the preparation of appropriately substituted 2-deoxy-1,4-dithio-D-erythro-pentofuranosides have proven problematic for large scale synthesis. To address these issues, herein we describe the modification and optimization of previously reported methods to allow for the convenient large scale synthesis of benzyl substituted 2-deoxy-1,4-dithio-D-erythro-pentofuranosides. Furthermore, we describe the development of reaction conditions for ß-selective glycosylation reactions of benzyl substituted 2-deoxy-1,4-dithio-D-erythro-pentofuranosides with both N4-benzoylcytosine and 5-aza-cytosine to enable the practical multi-gram syntheses of the clinical candidates 4'-thio-2'-deoxycytidine (T-dCyd) and 5-aza-4'-thio-2'-deoxycytidine (aza-T-dCyd). Taken together, these new synthetic developments have made possible the preclinical and early clinical development of these important anticancer agents at the National Cancer Institute.


Assuntos
Desoxicitidina/química , Desoxicitidina/síntese química , Tetroses/química , Técnicas de Química Sintética , Descoberta de Drogas , Glicosilação
12.
Glycoconj J ; 38(3): 347-359, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33245448

RESUMO

Proteins in the eye lens have negligible turnover and therefore progressively accumulate chemical modifications during aging. Carbonyls and oxidative stresses, which are intricately linked to one another, predominantly drive such modifications. Oxidative stress leads to the loss of glutathione (GSH) and ascorbate degradation; this in turn leads to the formation of highly reactive dicarbonyl compounds that react with proteins to form advanced glycation end products (AGEs). The formation of AGEs leads to the crosslinking and aggregation of proteins contributing to lens aging and cataract formation. To inhibit AGE formation, we developed a disulfide compound linking GSH diester and mercaptoethylguanidine, and we named it carboxitin. Bovine lens organ cultured with carboxitin showed higher levels of GSH and mercaptoethylguanidine in the lens nucleus. Carboxitin inhibited erythrulose-mediated mouse lens protein crosslinking, AGE formation and the formation of 3-deoxythreosone, a major ascorbate-derived AGE precursor in the human lens. Carboxitin inhibited the glycation-mediated increase in stiffness in organ-cultured mouse lenses measured using compressive mechanical strain. Delivery of carboxitin into the lens increases GSH levels, traps dicarbonyl compounds and inhibits AGE formation. These properties of carboxitin could be exploited to develop a therapy against the formation of AGEs and the increase in stiffness that causes presbyopia in aging lenses.


Assuntos
Glutationa/análogos & derivados , Glutationa/síntese química , Cristalino/efeitos dos fármacos , Animais , Bovinos , Produtos Finais de Glicação Avançada , Glicosilação , Cristalino/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Tetroses/metabolismo , Células Tumorais Cultivadas
13.
Anal Biochem ; 613: 114022, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33217405

RESUMO

In a recent paper, we showed the difference between the first stage of the one-substrate and the two-substrate transketolase reactions - the possibility of transfer of glycolaldehyde formed as a result of cleavage of the donor substrate from the thiazole ring of thiamine diphosphate to its aminopyrimidine ring through the tricycle formation stage, which is necessary for binding and splitting the second molecule of donor substrate [O.N. Solovjeva et al., The mechanism of a one-substrate transketolase reaction, Biosci. Rep. 40 (8) (2020) BSR20180246]. Here we show that under the action of the reducing agent a tricycle accumulates in a significant amount. Therefore, a significant decrease in the reaction rate of the one-substrate transketolase reaction compared to the two-substrate reaction is due to the stage of transferring the first glycolaldehyde molecule from the thiazole ring to the aminopyrimidine ring of thiamine diphosphate. Fragmentation of the four-carbon thiamine diphosphate derivatives showed that two glycolaldehyde molecules are bound to both coenzyme rings and the erythrulose molecule is bound to a thiazole ring. It was concluded that in the one-substrate reaction erythrulose is formed on the thiazole ring of thiamine diphosphate from two glycol aldehyde molecules linked to both thiamine diphosphate rings. The kinetic characteristics were determined for the two substrates, fructose 6-phosphate and glycolaldehyde.


Assuntos
Transcetolase/química , Transcetolase/metabolismo , Acetaldeído/análogos & derivados , Acetaldeído/química , Acetaldeído/metabolismo , Biocatálise , Boroidretos/química , Coenzimas/metabolismo , Frutosefosfatos/química , Frutosefosfatos/metabolismo , Cinética , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Especificidade por Substrato , Tetroses/metabolismo , Tiamina Pirofosfato/química , Tiamina Pirofosfato/metabolismo
14.
Nucleic Acids Res ; 49(2): 646-656, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33347562

RESUMO

The prebiotic synthesis of ribonucleotides is likely to have been accompanied by the synthesis of noncanonical nucleotides including the threo-nucleotide building blocks of TNA. Here, we examine the ability of activated threo-nucleotides to participate in nonenzymatic template-directed polymerization. We find that primer extension by multiple sequential threo-nucleotide monomers is strongly disfavored relative to ribo-nucleotides. Kinetic, NMR and crystallographic studies suggest that this is due in part to the slow formation of the imidazolium-bridged TNA dinucleotide intermediate in primer extension, and in part because of the greater distance between the attacking RNA primer 3'-hydroxyl and the phosphate of the incoming threo-nucleotide intermediate. Even a single activated threo-nucleotide in the presence of an activated downstream RNA oligonucleotide is added to the primer 10-fold more slowly than an activated ribonucleotide. In contrast, a single activated threo-nucleotide at the end of an RNA primer or in an RNA template results in only a modest decrease in the rate of primer extension, consistent with the minor and local structural distortions revealed by crystal structures. Our results are consistent with a model in which heterogeneous primordial oligonucleotides would, through cycles of replication, have given rise to increasingly homogeneous RNA strands.


Assuntos
Moldes Genéticos , Tetroses/química , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Origem da Vida , Polimerização , RNA/química , Relação Estrutura-Atividade
15.
Chem Commun (Camb) ; 56(93): 14653-14656, 2020 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-33155587

RESUMO

Threose nucleic acid (TNA) aptamers were selected in vitro to bind PD-L1 protein and inhibit its interaction with PD-1. These biologically stable TNA aptamers bound target proteins with nanomolar affinities, and effectively blocked PD-1/PD-L1 interaction in vitro. After injection into a colon cancer xenograft mouse model, the TNA aptamer N5 was specifically accumulated at the tumour site, and significantly inhibited tumour growth in vivo.


Assuntos
Aptâmeros de Nucleotídeos/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Imunoterapia , Neoplasias/terapia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Tetroses/química , Animais , Humanos , Camundongos
16.
J Am Chem Soc ; 142(41): 17766-17781, 2020 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-33017148

RESUMO

Controlling the structure and activity of nucleic acids dramatically expands their potential for application in therapeutics, biosensing, nanotechnology, and biocomputing. Several methods have been developed to impart responsiveness of DNA and RNA to small-molecule and light-based stimuli. However, heat-triggered control of nucleic acids has remained largely unexplored, leaving a significant gap in responsive nucleic acid technology. Moreover, current technologies have been limited to natural nucleic acids and are often incompatible with polymerase-generated sequences. Here we show that glyoxal, a well-characterized compound that covalently attaches to the Watson-Crick-Franklin face of several nucleobases, addresses these limitations by thermoreversibly modulating the structure and activity of virtually any nucleic acid scaffold. Using a variety of DNA and RNA constructs, we demonstrate that glyoxal modification is easily installed and potently disrupts nucleic acid structure and function. We also characterize the kinetics of decaging and show that activity can be restored via tunable thermal removal of glyoxal adducts under a variety of conditions. We further illustrate the versatility of this approach by reversibly caging a 2'-O-methylated RNA aptamer as well as synthetic threose nucleic acid (TNA) and peptide nucleic acid (PNA) scaffolds. Glyoxal caging can also be used to reversibly disrupt enzyme-nucleic acid interactions, and we show that caging of guide RNA allows for tunable and reversible control over CRISPR-Cas9 activity. We also demonstrate glyoxal caging as an effective method for enhancing PCR specificity, and we cage a biostable antisense oligonucleotide for time-release activation and titration of gene expression in living cells. Together, glyoxalation is a straightforward and scarless method for imparting reversible thermal responsiveness to theoretically any nucleic acid architecture, addressing a significant need in synthetic biology and offering a versatile new tool for constructing programmable nucleic acid components in medicine, nanotechnology, and biocomputing.


Assuntos
Glioxal/química , Ácidos Nucleicos/química , Sequência de Bases , Catálise , Domínio Catalítico , Metilação , Conformação de Ácido Nucleico , Oligonucleotídeos/química , Ácidos Nucleicos Peptídicos/química , Relação Estrutura-Atividade , Biologia Sintética , Tetroses/química , Termodinâmica
17.
Molecules ; 25(18)2020 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-32933142

RESUMO

Recent advances in polymerase engineering have made it possible to isolate aptamers from libraries of synthetic genetic polymers (XNAs) with backbone structures that are distinct from those found in nature. However, nearly all of the XNA aptamers produced thus far have been generated against protein targets, raising significant questions about the ability of XNA aptamers to recognize small molecule targets. Here, we report the evolution of an ATP-binding aptamer composed entirely of α-L-threose nucleic acid (TNA). A chemically synthesized version of the best aptamer sequence shows high affinity to ATP and strong specificity against other naturally occurring ribonucleotide triphosphates. Unlike its DNA and RNA counterparts that are susceptible to nuclease digestion, the ATP-binding TNA aptamer exhibits high biological stability against hydrolytic enzymes that rapidly degrade DNA and RNA. Based on these findings, we suggest that TNA aptamers could find widespread use as molecular recognition elements in diagnostic and therapeutic applications that require high biological stability.


Assuntos
Trifosfato de Adenosina/química , Aptâmeros de Nucleotídeos/química , Oligonucleotídeos/química , Bibliotecas de Moléculas Pequenas/química , Tetroses/química , Sequência de Bases , Engenharia Genética , Conformação de Ácido Nucleico , Ribonucleotídeos/química , Técnica de Seleção de Aptâmeros , Técnicas de Síntese em Fase Sólida
18.
Chem Commun (Camb) ; 56(68): 9894-9897, 2020 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-32720666

RESUMO

A terminal-closed linear gene with strong exonuclease resistance and serum stability was successfully constructed by polymerase chain reaction (PCR) with an α-l-threose nucleic acid (TNA) loop modified primer pair, which can be used as an efficient gene expression system in eukaryotic cells for gene delivery.


Assuntos
Primers do DNA/química , Ácidos Nucleicos/química , Tetroses/química , Transfecção/métodos , Primers do DNA/metabolismo , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Microscopia de Fluorescência , Plasmídeos/genética , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase
19.
J Am Chem Soc ; 142(9): 4379-4389, 2020 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-32023044

RESUMO

This paper reports an investigation into organocatalytic hydrogels as prebiotically relevant systems. Gels are interesting prebiotic reaction media, combining heterogeneous and homogeneous characteristics with a structurally organized active "solid-like" catalyst separated from the surrounding environment, yet in intimate contact with the solution phase and readily accessible via "liquid-like" diffusion. A simple self-assembling glutamine amide derivative 1 was initially found to catalyze a model aldol reaction between cyclohexanone and 4-nitrobenzaldehyde, but it did not maintain its gel structure during reaction. In this study, it was observed that compound 1 could react directly with the benzaldehyde to form a hydrogel in situ based on Schiff base 2 as a low-molecular-weight gelator (LMWG). This new dynamic gel is a rare example of a two-component self-assembled LMWG hydrogel and was fully characterized. It was demonstrated that glutamine amide 1 could select an optimal aldehyde component and preferentially assemble from mixtures. In the hunt for an organocatalyst, reductive conditions were applied to the Schiff base to yield secondary amine 3, which is also a highly effective hydrogelator at very low loadings with a high degree of nanoscale order. Most importantly, the hydrogel based on 3 catalyzed the prebiotically relevant aldol dimerization of glycolaldehyde to give threose and erythrose. In buffered conditions, this reaction gave excellent conversions, good diastereoselectivity, and some enantioselectivity. Catalysis using the hydrogel of 3 was much better than that using non-assembled 3-demonstrating a clear benefit of self-assembly. The results suggest that hydrogels offer a potential strategy by which prebiotic reactions can be promoted using simple, prebiotically plausible LMWGs that can selectively self-organize from complex mixtures. Such processes may have been of prebiotic importance.


Assuntos
Glutamina/análogos & derivados , Hidrogéis/química , Acetaldeído/análogos & derivados , Acetaldeído/química , Benzaldeídos/química , Catálise , Cicloexanonas/química , Bases de Schiff/síntese química , Estereoisomerismo , Tetroses/síntese química
20.
Cell Biol Int ; 44(2): 651-660, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31750586

RESUMO

In response to osmotic stress, the yeast Yarrowia lipolytica produces erythritol, a four-carbon sugar alcohol, from erythrose-P, an intermediate of the pentose phosphate pathway. Under non-stressing conditions (isotonic environment), the produced erythritol is subsequently recycled into erythrose-P that can feed the pentose phosphate pathway. Herein, gene YALI0F01584g was characterized as involved in the erythritol catabolic pathway. Several experimental evidences suggested that it encodes an erythrulose-1P isomerase that converts erythrulose-1P into erythrulose-4P. On the basis of our previous reports and results gathered in this study with genetically modified strains, including ΔYALI0F01584g and ΔYALI0F01628g disrupted mutants, the entire erythritol catabolic pathway has been characterized.


Assuntos
Eritritol/metabolismo , Proteínas Fúngicas/metabolismo , Fosfatos/metabolismo , Tetroses/metabolismo , Yarrowia/metabolismo , Sequência de Aminoácidos , Proteínas Fúngicas/genética , Homologia de Sequência , Yarrowia/genética , Yarrowia/crescimento & desenvolvimento
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