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1.
ChemistryOpen ; 13(8): e202300232, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38200655

RESUMO

We recently reported the properties of RNA hairpins constrained by a dimethylene (DME) disulfide (S-S) linker incorporated between two adjacent nucleosides in the loop and showed that this linker locked the hairpin conformation thus disturbing the duplex/hairpin equilibrium. We have now investigated the influence of the length of the linker and synthesized oligoribonucleotides containing diethylene (DEE) and dipropylene (DPE) S-S bridges. This was achieved via the preparation of building blocks, namely 2'-O-acetylthioethyl (2'-O-AcSE) and 2'-O-acetylthiopropyl (2'-O-AcSP) uridine phosphoramidites, which were successfully incorporated into RNA sequences. Thermal denaturation analysis revealed that the DEE and DPE disulfide bridges destabilize RNA duplexes but do not disrupt the hairpin conformation. Furthermore, our investigation of the duplex/hairpin equilibrium indicated that sequences modified with DME and DEE S-S linkers predominantly lock the hairpin form, whereas the DPE S-S linker provides flexibility. These findings highlight the potential of S-S linkers to study RNA interactions.


Assuntos
Dissulfetos , Conformação de Ácido Nucleico , RNA , RNA/química , Dissulfetos/química , Desnaturação de Ácido Nucleico , Oligorribonucleotídeos/química , Oligorribonucleotídeos/síntese química , Compostos Organofosforados/química , Uridina/química , Uridina/análogos & derivados
2.
Int J Mol Sci ; 24(21)2023 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-37958961

RESUMO

A template-assisted assembly approach to a C24 fullerene-like double-stranded DNA polyhedral shell is proposed. The assembly employed a supramolecular oligonucleotide dendrimer as a 3D template that was obtained via the hybridization of siRNA strands and a single-stranded DNA oligonucleotide joined to three- or four-way branched junctions. A four-way branched oligonucleotide building block (a starlet) was designed for the assembly of the shell composed of three identical self-complementary DNA single strands and a single RNA strand for hybridization to the DNA oligonucleotides of the template. To prevent premature auto-hybridization of the self-complementary oligonucleotides in the starlet, a photolabile protecting group was introduced via the N3-substituted thymidine phosphoramidite. Cleavable linkers such as a disulfide linkage, RNase A sensitive triribonucleotides, and di- and trideoxynucleotides were incorporated into the starlet and template at specific points to guide the post-assembly disconnection of the shell from the template, and enzymatic disassembly of the template and the shell in biological media. At the same time, siRNA strands were modified with 2'-OMe ribonucleotides and phosphorothioate groups in certain positions to stabilize toward enzymatic digestion. We report herein a solid-phase synthesis of branched oligodeoxy and oligoribonucleotide building blocks for the DNA/RNA dendritic template and the branched DNA starlet for a template-assisted construction of a C24 fullerene-like DNA shell after initial molecular modeling, followed by the assembly of the shell around the DNA-coated RNA dendritic template, and visualization of the resulting nanostructure by transmission electron microscopy.


Assuntos
Fulerenos , Nanoestruturas , Oligorribonucleotídeos/química , DNA/química , Nanoestruturas/química , Oligonucleotídeos/química , RNA Interferente Pequeno , Conformação de Ácido Nucleico
3.
Nucleic Acids Res ; 50(19): 11199-11213, 2022 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-36271789

RESUMO

Standalone ring nucleases are CRISPR ancillary proteins, which downregulate the immune response of Type III CRISPR-Cas systems by cleaving cyclic oligoadenylates (cA) second messengers. Two genes with this function have been found within the Sulfolobus islandicus (Sis) genome. They code for a long polypeptide composed by a CARF domain fused to an HTH domain and a short polypeptide constituted by a CARF domain with a 40 residue C-terminal insertion. Here, we determine the structure of the apo and substrate bound states of the Sis0455 enzyme, revealing an insertion at the C-terminal region of the CARF domain, which plays a key role closing the catalytic site upon substrate binding. Our analysis reveals the key residues of Sis0455 during cleavage and the coupling of the active site closing with their positioning to proceed with cA4 phosphodiester hydrolysis. A time course comparison of cA4 cleavage between the short, Sis0455, and long ring nucleases, Sis0811, shows the slower cleavage kinetics of the former, suggesting that the combination of these two types of enzymes with the same function in a genome could be an evolutionary strategy to regulate the levels of the second messenger in different infection scenarios.


Assuntos
Proteínas Associadas a CRISPR , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Oligorribonucleotídeos/química , Nucleotídeos de Adenina/metabolismo , Endonucleases/metabolismo
4.
J Am Soc Mass Spectrom ; 33(3): 510-520, 2022 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-35157441

RESUMO

With the increased development of new RNA-based therapeutics, the need for robust analytical methods for confirming sequences and mapping modifications has accelerated. Characterizing modified ribonucleic acids using mass spectrometry is challenging because diagnostic fragmentation may be suppressed for modified nucleotides, thus hampering complete sequence coverage and the confident localization of modifications. Ultraviolet photodissociation (UVPD) has shown great potential for the characterization of nucleic acids due to extensive backbone fragmentation. Activated electron photodetachment dissociation (a-EPD) has also been used as an alternative to capitalize on the dominant charge-reduction pathway prevalent in UVPD, facilitate dissociation, and produce high abundances of fragment ions. Here, we compare higher-energy collisional activation (HCD), UVPD using 193 and 213 nm photons, and a-EPD for the top-down sequencing of modified nucleic acids, including methylated, phosphorothioate, and locked nucleic acid-modified DNA. The presence of these modifications alters the fragmentation pathways observed upon UVPD and a-EPD, and extensive backbone cleavage is observed that results in the production of fragment ions that retain the modifications and allow them to be pinpointed. LNA and 2'-O-methoxy phosphorothioate modifications caused a significant suppression of fragmentation for UVPD but not for a-EPD, whereas phosphorothioate bonds did not cause any significant suppression for either method. The incorporation of 2'-O-methyl modifications suppressed fragmentation of the antisense strand of patisiran, which resulted in some gaps in sequence coverage. However, UVPD provided the highest sequence coverage when compared to a-EPD.


Assuntos
Espectrometria de Massas/métodos , Oligorribonucleotídeos , Análise de Sequência/métodos , Elétrons , Oligorribonucleotídeos/análise , Oligorribonucleotídeos/química , Oligorribonucleotídeos/efeitos da radiação , Fotólise , Raios Ultravioleta
5.
Bioorg Med Chem ; 56: 116632, 2022 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-35078032

RESUMO

The oligoadenylate synthetase-ribonuclease L pathway is a major player in the interferon-induced antiviral defense mechanism of cells. Upon sensing viral dsRNA, 5'-phosphorylated 2',5'-oligoadenylates are synthesized, and subsequently activate latent RNase L. To determine the influence of 5'-phosphate end on the activation of human RNase L, four sets of 5'-phosphonate modified oligoadenylates were prepared on solid-phase. The ability of these 5'-modified oligoadenylates bearing shortened, isosteric and prolonged phosphonate linkages to activate RNase L was explored. We found that isosteric linkages and linkages prolonged by one atom were in general well tolerated by the enzyme with the EC50 values comparable to that of the natural activator. In contrast, linkages shortened by one atom or prolonged by two atoms exhibited decrease in the activity.


Assuntos
Nucleotídeos de Adenina/farmacologia , Endorribonucleases/metabolismo , Oligorribonucleotídeos/farmacologia , Organofosfonatos/farmacologia , Nucleotídeos de Adenina/síntese química , Nucleotídeos de Adenina/química , Relação Dose-Resposta a Droga , Humanos , Conformação de Ácido Nucleico , Oligorribonucleotídeos/síntese química , Oligorribonucleotídeos/química , Organofosfonatos/síntese química , Organofosfonatos/química , Relação Estrutura-Atividade
6.
J Biol Chem ; 298(2): 101591, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35038453

RESUMO

RNA interference by type III CRISPR systems results in the synthesis of cyclic oligoadenylate (cOA) second messengers, which are known to bind and regulate various CARF domain-containing nuclease receptors. The CARF domain-containing Csa3 family of transcriptional factors associated with the DNA-targeting type I CRISPR systems regulate expression of various CRISPR and DNA repair genes in many prokaryotes. In this study, we extend the known receptor repertoire of cOA messengers to include transcriptional factors by demonstrating specific binding of cyclic tetra-adenylate (cA4) to Saccharolobus solfataricus Csa3 (Csa3Sso). Our 2.0-Å resolution X-ray crystal structure of cA4-bound full-length Csa3Sso reveals the binding of its CARF domain to an elongated conformation of cA4. Using cA4 binding affinity analyses of Csa3Sso mutants targeting the observed Csa3Sso•cA4 structural interface, we identified a Csa3-specific cA4 binding motif distinct from a more widely conserved cOA-binding CARF motif. Using a rational surface engineering approach, we increased the cA4 binding affinity of Csa3Sso up to ∼145-fold over the wildtype, which has potential applications for future second messenger-driven CRISPR gene expression and editing systems. Our in-solution Csa3Sso structural analysis identified cA4-induced allosteric and asymmetric conformational rearrangement of its C-terminal winged helix-turn-helix effector domains, which could potentially be incompatible to DNA binding. However, specific in vitro binding of the purified Csa3Sso to its putative promoter (PCas4a) was found to be cA4 independent, suggesting a complex mode of Csa3Sso regulation. Overall, our results support cA4-and Csa3-mediated cross talk between type III and type I CRISPR systems.


Assuntos
Nucleotídeos de Adenina , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Oligorribonucleotídeos , Nucleotídeos de Adenina/química , Nucleotídeos de Adenina/metabolismo , Sistemas CRISPR-Cas , DNA/genética , Modelos Moleculares , Oligorribonucleotídeos/química , Oligorribonucleotídeos/metabolismo , Relação Estrutura-Atividade , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
7.
Nucleic Acids Res ; 49(21): 12577-12590, 2021 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-34850143

RESUMO

Type III CRISPR-Cas effector systems detect foreign RNA triggering DNA and RNA cleavage and synthesizing cyclic oligoadenylate molecules (cA) in their Cas10 subunit. cAs act as a second messenger activating auxiliary nucleases, leading to an indiscriminate RNA degradation that can end in cell dormancy or death. Standalone ring nucleases are CRISPR ancillary proteins which downregulate the strong immune response of Type III systems by degrading cA. These enzymes contain a CRISPR-associated Rossman-fold (CARF) domain, which binds and cleaves the cA molecule. Here, we present the structures of the standalone ring nuclease from Sulfolobus islandicus (Sis) 0811 in its apo and post-catalytic states. This enzyme is composed by a N-terminal CARF and a C-terminal wHTH domain. Sis0811 presents a phosphodiester hydrolysis metal-independent mechanism, which cleaves cA4 rings to generate linear adenylate species, thus reducing the levels of the second messenger and switching off the cell antiviral state. The structural and biochemical analysis revealed the coupling of a cork-screw conformational change with the positioning of key catalytic residues to proceed with cA4 phosphodiester hydrolysis in a non-concerted manner.


Assuntos
Nucleotídeos de Adenina/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Endonucleases/metabolismo , Nucleotídeos Cíclicos/metabolismo , Oligorribonucleotídeos/metabolismo , Sulfolobus solfataricus/enzimologia , Nucleotídeos de Adenina/química , Sítios de Ligação/genética , Biocatálise , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/genética , Cromatografia Líquida , Cristalografia por Raios X , Endonucleases/química , Endonucleases/genética , Cinética , Espectrometria de Massas/métodos , Modelos Moleculares , Mutação , Nucleotídeos Cíclicos/química , Oligorribonucleotídeos/química , Domínios Proteicos , Sulfolobus solfataricus/genética
8.
Nucleic Acids Res ; 49(19): 10851-10867, 2021 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-34648028

RESUMO

We recently reported that RNAi-mediated off-target effects are important drivers of the hepatotoxicity observed for a subset of GalNAc-siRNA conjugates in rodents, and that these findings could be mitigated by seed-pairing destabilization using a single GNA nucleotide placed within the seed region of the guide strand. Here, we report further investigation of the unique and poorly understood GNA/RNA cross-pairing behavior to better inform GNA-containing siRNA design. A reexamination of published GNA homoduplex crystal structures, along with a novel structure containing a single (S)-GNA-A residue in duplex RNA, indicated that GNA nucleotides universally adopt a rotated nucleobase orientation within all duplex contexts. Such an orientation strongly affects GNA-C and GNA-G but not GNA-A or GNA-T pairing in GNA/RNA heteroduplexes. Transposition of the hydrogen-bond donor/acceptor pairs using the novel (S)-GNA-isocytidine and -isoguanosine nucleotides could rescue productive base-pairing with the complementary G or C ribonucleotides, respectively. GalNAc-siRNAs containing these GNA isonucleotides showed an improved in vitro activity, a similar improvement in off-target profile, and maintained in vivo activity and guide strand liver levels more consistent with the parent siRNAs than those modified with isomeric GNA-C or -G, thereby expanding our toolbox for the design of siRNAs with minimized off-target activity.


Assuntos
Adenosina/química , Citidina/química , Glicóis/química , Guanosina/química , Oligorribonucleotídeos/química , RNA de Cadeia Dupla/química , RNA Interferente Pequeno/química , Acetilgalactosamina , Oxirredutases do Álcool/antagonistas & inibidores , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Animais , Pareamento de Bases , Células COS , Chlorocebus aethiops , Dimetilformamida/análogos & derivados , Dimetilformamida/química , Etilaminas/química , Feminino , Hepatócitos/citologia , Hepatócitos/metabolismo , Ligação de Hidrogênio , Camundongos , Camundongos Endogâmicos C57BL , Oligorribonucleotídeos/genética , Oligorribonucleotídeos/metabolismo , Compostos Organofosforados/química , Pré-Albumina/antagonistas & inibidores , Pré-Albumina/genética , Pré-Albumina/metabolismo , Cultura Primária de Células , Estabilidade de RNA , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo
9.
Curr Issues Mol Biol ; 43(3): 1267-1281, 2021 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-34698059

RESUMO

Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease caused by out-of-frame or nonsense mutation in the dystrophin gene. It begins with a loss of ambulation between 9 and 14 years of age, followed by various other symptoms including cardiac dysfunction. Exon skipping of patients' DMD pre-mRNA induced by antisense oligonucleotides (AOs) is expected to produce shorter but partly functional dystrophin proteins, such as those possessed by patients with the less severe Becker muscular dystrophy. We are working on developing modified nucleotides, such as 2'-O,4'-C-ethylene-bridged nucleic acids (ENAs), possessing high nuclease resistance and high affinity for complementary RNA strands. Here, we demonstrate the preclinical characteristics (exon-skipping activity in vivo, stability in blood, pharmacokinetics, and tissue distribution) of renadirsen, a novel AO modified with 2'-O-methyl RNA/ENA chimera phosphorothioate designed for dystrophin exon 45 skipping and currently under clinical trials. Notably, systemic delivery of renadirsen sodium promoted dystrophin exon skipping in cardiac muscle, skeletal muscle, and diaphragm, compared with AOs with the same sequence as renadirsen but conventionally modified by PMO and 2'OMePS. These findings suggest the promise of renadirsen sodium as a therapeutic agent that improves not only skeletal muscle symptoms but also other symptoms in DMD patients, such as cardiac dysfunction.


Assuntos
Processamento Alternativo , Distrofina/genética , Oligonucleotídeos Antissenso/genética , Animais , Cromatografia Líquida , Masculino , Camundongos , Camundongos Endogâmicos mdx , Estrutura Molecular , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Oligodesoxirribonucleotídeos/química , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/síntese química , Oligonucleotídeos Antissenso/química , Oligorribonucleotídeos/química , Espectrometria de Massas em Tandem , Distribuição Tecidual
10.
Nat Commun ; 12(1): 5033, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34413302

RESUMO

Characteristic properties of type III CRISPR-Cas systems include recognition of target RNA and the subsequent induction of a multifaceted immune response. This involves sequence-specific cleavage of the target RNA and production of cyclic oligoadenylate (cOA) molecules. Here we report that an exposed seed region at the 3' end of the crRNA is essential for target RNA binding and cleavage, whereas cOA production requires base pairing at the 5' end of the crRNA. Moreover, we uncover that the variation in the size and composition of type III complexes within a single host results in variable seed regions. This may prevent escape by invading genetic elements, while controlling cOA production tightly to prevent unnecessary damage to the host. Lastly, we use these findings to develop a new diagnostic tool, SCOPE, for the specific detection of SARS-CoV-2 from human nasal swab samples, revealing sensitivities in the atto-molar range.


Assuntos
Nucleotídeos de Adenina/química , COVID-19/diagnóstico , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Oligorribonucleotídeos/química , RNA Bacteriano/genética , Ribonucleases/metabolismo , SARS-CoV-2/genética , COVID-19/genética , COVID-19/metabolismo , COVID-19/virologia , Testes Diagnósticos de Rotina/métodos , Humanos , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/patogenicidade
11.
Nucleic Acids Res ; 48(18): 10087-10100, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-32941619

RESUMO

The N4-methylation of cytidine (m4C and m42C) in RNA plays important roles in both bacterial and eukaryotic cells. In this work, we synthesized a series of m4C and m42C modified RNA oligonucleotides, conducted their base pairing and bioactivity studies, and solved three new crystal structures of the RNA duplexes containing these two modifications. Our thermostability and X-ray crystallography studies, together with the molecular dynamic simulation studies, demonstrated that m4C retains a regular C:G base pairing pattern in RNA duplex and has a relatively small effect on its base pairing stability and specificity. By contrast, the m42C modification disrupts the C:G pair and significantly decreases the duplex stability through a conformational shift of native Watson-Crick pair to a wobble-like pattern with the formation of two hydrogen bonds. This double-methylated m42C also results in the loss of base pairing discrimination between C:G and other mismatched pairs like C:A, C:T and C:C. The biochemical investigation of these two modified residues in the reverse transcription model shows that both mono- or di-methylated cytosine bases could specify the C:T pair and induce the G to T mutation using HIV-1 RT. In the presence of other reverse transcriptases with higher fidelity like AMV-RT, the methylation could either retain the normal nucleotide incorporation or completely inhibit the DNA synthesis. These results indicate the methylation at N4-position of cytidine is a molecular mechanism to fine tune base pairing specificity and affect the coding efficiency and fidelity during gene replication.


Assuntos
Pareamento de Bases , Citidina/química , Oligorribonucleotídeos/química , RNA/química , Metilação , Oligorribonucleotídeos/síntese química , Dobramento de RNA
12.
Curr Protoc Nucleic Acid Chem ; 82(1): e114, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32846053

RESUMO

This article contains detailed synthetic protocols for preparation of 5-cyanomethyluridine (cnm5 U) and 5-cyanouridine (cn5 U) phosphoramidites. The synthesis of the cnm5 U phosphoramidite building block starts with commercially available 5-methyluridine (m5 C), followed by bromination of the 5-methyl group to install the cyano moiety using TMSCN/TBAF. The cn5 U phosphoramidite is obtained by regular Vorbrüggen glycosylation of the protected ribofuranose with silylated 5-cyanouracil. These two modified phosphoramidites are suitable for synthesis of RNA oligonucleotides on solid phase using conventional amidite chemistry. Our protocol provides access to two novel building blocks for constructing RNA-based therapeutics. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Preparation of cnm5 U and cn5 U phosphoramidites Basic Protocol 2: Synthesis, purification, and characterization of cnm5 U- and cn5 U-modified RNA oligonucleotides.


Assuntos
Cianetos/química , Oligorribonucleotídeos/química , Compostos Organofosforados/síntese química , Cromatografia em Camada Fina , Oligorribonucleotídeos/síntese química , Oligorribonucleotídeos/isolamento & purificação , Compostos Organofosforados/química , Espectroscopia de Prótons por Ressonância Magnética , Técnicas de Síntese em Fase Sólida/métodos , Espectrometria de Massas por Ionização por Electrospray
13.
Molecules ; 25(15)2020 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-32717917

RESUMO

The chemical synthesis of modified oligoribonucleotides represents a powerful approach to study the structure, stability, and biological activity of RNAs. Selected RNA modifications have been proven to enhance the drug-like properties of RNA oligomers providing the oligonucleotide-based therapeutic agents in the antisense and siRNA technologies. The important sites of RNA modification/functionalization are the nucleobase residues. Standard phosphoramidite RNA chemistry allows the site-specific incorporation of a large number of functional groups to the nucleobase structure if the building blocks are synthetically obtainable and stable under the conditions of oligonucleotide chemistry and work-up. Otherwise, the chemically modified RNAs are produced by post-synthetic oligoribonucleotide functionalization. This review highlights the post-synthetic RNA modification approach as a convenient and valuable method to introduce a wide variety of nucleobase modifications, including recently discovered native hypermodified functional groups, fluorescent dyes, photoreactive groups, disulfide crosslinks, and nitroxide spin labels.


Assuntos
Oligorribonucleotídeos/síntese química , Compostos Organofosforados/química , RNA/química , Modelos Moleculares , Conformação de Ácido Nucleico , Oligorribonucleotídeos/química , Estabilidade de RNA
14.
Int J Mol Sci ; 21(14)2020 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-32698484

RESUMO

We report a universal straightforward strategy for the chemical synthesis of modified oligoribonucleotides containing functional groups of different structures at the 2' position of ribose. The on-column synthetic concept is based on the incorporation of two types of commercial nucleotide phosphoramidites containing orthogonal 2'-O-protecting groups, namely 2'-O-thiomorpholine-carbothioate (TC, as "permanent") and 2'-O-tert-butyl(dimethyl)silyl (tBDMS, as "temporary"), to RNA during solid-phase synthesis. Subsequently, the support-bound RNA undergoes selective deprotection and follows postsynthetic 2' functionalization of the naked hydroxyl group. This convenient method to tailor RNA, utilizing the advantages of solid phase approaches, gives an opportunity to introduce site-specifically a wide range of linkers and functional groups. By this strategy, a series of RNAs containing diverse 2' functionalities were synthesized and studied with respect to their physicochemical properties.


Assuntos
Oligorribonucleotídeos/síntese química , RNA/síntese química , Técnicas de Síntese em Fase Sólida/métodos , Sequência de Bases , Desnaturação de Ácido Nucleico , Oligorribonucleotídeos/química , Compostos Organofosforados/síntese química , Compostos Organofosforados/química , RNA/química
15.
Nucleic Acids Res ; 48(14): 7640-7652, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32558908

RESUMO

With the central role of nucleic acids there is a need for development of fluorophores that facilitate the visualization of processes involving nucleic acids without perturbing their natural properties and behaviour. Here, we incorporate a new analogue of adenine, 2CNqA, into both DNA and RNA, and evaluate its nucleobase-mimicking and internal fluorophore capacities. We find that 2CNqA displays excellent photophysical properties in both nucleic acids, is highly specific for thymine/uracil, and maintains and slightly stabilises the canonical conformations of DNA and RNA duplexes. Moreover, the 2CNqA fluorophore has a quantum yield in single-stranded and duplex DNA ranging from 10% to 44% and 22% to 32%, respectively, and a slightly lower one (average 12%) inside duplex RNA. In combination with a comparatively strong molar absorptivity for this class of compounds, the resulting brightness of 2CNqA inside double-stranded DNA is the highest reported for a fluorescent base analogue. The high, relatively sequence-independent quantum yield in duplexes makes 2CNqA promising as a nucleic acid label and as an interbase Förster resonance energy transfer (FRET) donor. Finally, we report its excellent spectral overlap with the interbase FRET acceptors qAnitro and tCnitro, and demonstrate that these FRET pairs enable conformation studies of DNA and RNA.


Assuntos
DNA/química , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , RNA de Cadeia Dupla/química , Pareamento de Bases , DNA de Cadeia Simples/química , Oligodesoxirribonucleotídeos/síntese química , Oligodesoxirribonucleotídeos/química , Oligorribonucleotídeos/síntese química , Oligorribonucleotídeos/química
16.
Nucleic Acids Res ; 48(14): e80, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32496547

RESUMO

Small RNAs are important regulators of gene expression and are involved in human development and disease. Next generation sequencing (NGS) allows for scalable, genome-wide studies of small RNA; however, current methods are challenged by low sensitivity and high bias, limiting their ability to capture an accurate representation of the cellular small RNA population. Several studies have shown that this bias primarily arises during the ligation of single-strand adapters during library preparation, and that this ligation bias is magnified by 2'-O-methyl modifications (2'OMe) on the 3' terminal nucleotide. In this study, we developed a novel library preparation process using randomized splint ligation with a cleavable adapter, a design which resolves previous challenges associated with this ligation strategy. We show that a randomized splint ligation based workflow can reduce bias and increase the sensitivity of small RNA sequencing for a wide variety of small RNAs, including microRNA (miRNA) and tRNA fragments as well as 2'OMe modified RNA, including Piwi-interacting RNA and plant miRNA. Finally, we demonstrate that this workflow detects more differentially expressed miRNA between tumorous and matched normal tissues. Overall, this library preparation process allows for highly accurate small RNA sequencing and will enable studies of 2'OMe modified RNA with new levels of detail.


Assuntos
Biblioteca Gênica , Pequeno RNA não Traduzido/isolamento & purificação , Análise de Sequência de RNA/métodos , Eletroforese Capilar , Feminino , Humanos , Masculino , Metilação , MicroRNAs/química , MicroRNAs/genética , MicroRNAs/isolamento & purificação , Hibridização de Ácido Nucleico , Oligorribonucleotídeos/química , RNA Neoplásico/química , RNA Neoplásico/genética , RNA Neoplásico/isolamento & purificação , RNA de Plantas/química , RNA de Plantas/genética , RNA de Plantas/isolamento & purificação , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/genética , RNA de Transferência/química , RNA de Transferência/isolamento & purificação , Distribuição Aleatória , Sensibilidade e Especificidade , Alinhamento de Sequência
17.
Bioorg Chem ; 99: 103815, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32289587

RESUMO

The development of novel delivery systems capable of enhancing the antibody binding affinity and immunoactivity of short length saccharide antigens is at the forefront of modern medicine. In this regard, gold nanoparticles (AuNPs) raised great interest as promising nano-vaccine platform, as they do not interfere with the desired immune response and their surface can be easily functionalized, enabling the antigen multivalent presentation. In addition, the nanoparticles morphology can have a great impact on their biological properties. Gram-positive Group A Streptococcus (GAS) is a bacterium responsible for many infections and represents a priority healthcare concern, but a universal vaccine is still unavailable. Since all the GAS strains have a cell wall characterized by a common polyrhamnose backbone, this can be employed as alternative antigen to develop an anti-GAS vaccine. Herein, we present the synthesis of two oligorhamnoside fragments and their corresponding oligorhamnoside-AuNPs, designed with two different morphologies. By competitive ELISA we assessed that both symmetric and anisotropic oligorhamnan nanoparticles inhibit the binding of specific polyclonal serum much better than the unconjugated oligosaccharides.


Assuntos
Anticorpos/imunologia , Ouro/química , Nanopartículas Metálicas/química , Oligorribonucleotídeos/imunologia , Streptococcus/química , Anticorpos/química , Configuração de Carboidratos , Ouro/imunologia , Oligorribonucleotídeos/síntese química , Oligorribonucleotídeos/química , Streptococcus/imunologia
18.
Nat Commun ; 11(1): 1596, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32221291

RESUMO

Bacterial and archaeal CRISPR-Cas systems provide RNA-guided immunity against genetic invaders such as bacteriophages and plasmids. Upon target RNA recognition, type III CRISPR-Cas systems produce cyclic-oligoadenylate second messengers that activate downstream effectors, including Csm6 ribonucleases, via their CARF domains. Here, we show that Enteroccocus italicus Csm6 (EiCsm6) degrades its cognate cyclic hexa-AMP (cA6) activator, and report the crystal structure of EiCsm6 bound to a cA6 mimic. Our structural, biochemical, and in vivo functional assays reveal how cA6 recognition by the CARF domain activates the Csm6 HEPN domains for collateral RNA degradation, and how CARF domain-mediated cA6 cleavage provides an intrinsic off-switch to limit Csm6 activity in the absence of ring nucleases. These mechanisms facilitate rapid invader clearance and ensure termination of CRISPR interference to limit self-toxicity.


Assuntos
Nucleotídeos de Adenina/química , Nucleotídeos de Adenina/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Endonucleases/química , Endonucleases/metabolismo , Oligorribonucleotídeos/química , Oligorribonucleotídeos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sistemas CRISPR-Cas , Cristalografia por Raios X , Ativação Enzimática , Modelos Moleculares , Domínios Proteicos , Estabilidade de RNA
19.
Angew Chem Int Ed Engl ; 59(26): 10645-10650, 2020 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-32198805

RESUMO

N6 -isopentenyladenosine (i6 A) is an RNA modification found in cytokinins, which regulate plant growth/differentiation, and a subset of tRNAs, where it improves the efficiency and accuracy of translation. The installation and removal of this modification is mediated by prenyltransferases and cytokinin oxidases, and a chemical approach to selective deprenylation of i6 A has not been developed. We show that a selected group of oxoammonium cations function as artificial deprenylases to promote highly selective deprenylation of i6 A in nucleosides, oligonucleotides, and live cells. Importantly, other epigenetic modifications, amino acid residues, and natural products were not affected. Moreover, a significant phenotype difference in the Arabidopsis thaliana shoot and root development was observed with incubation of the cation. These results establish these small organic molecules as direct chemical regulators/artificial deprenylases of i6 A.


Assuntos
Óxidos N-Cíclicos/farmacologia , Citocininas/metabolismo , Isopenteniladenosina/metabolismo , Piperidinas/farmacologia , Prenilação/efeitos dos fármacos , RNA/metabolismo , Arabidopsis/efeitos dos fármacos , Óxidos N-Cíclicos/química , Óxidos N-Cíclicos/toxicidade , Citocininas/química , Epigênese Genética/efeitos dos fármacos , Humanos , Isopenteniladenosina/química , Células MCF-7 , Oligorribonucleotídeos/química , Oligorribonucleotídeos/metabolismo , Piperidinas/química , Piperidinas/toxicidade , Reguladores de Crescimento de Plantas/química , Reguladores de Crescimento de Plantas/metabolismo , Raízes de Plantas/efeitos dos fármacos , Brotos de Planta/efeitos dos fármacos , RNA/química
20.
Nucleic Acids Res ; 48(7): 3734-3746, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32095818

RESUMO

Reverse transcription (RT) of RNA templates containing RNA modifications leads to synthesis of cDNA containing information on the modification in the form of misincorporation, arrest, or nucleotide skipping events. A compilation of such events from multiple cDNAs represents an RT-signature that is typical for a given modification, but, as we show here, depends also on the reverse transcriptase enzyme. A comparison of 13 different enzymes revealed a range of RT-signatures, with individual enzymes exhibiting average arrest rates between 20 and 75%, as well as average misincorporation rates between 30 and 75% in the read-through cDNA. Using RT-signatures from individual enzymes to train a random forest model as a machine learning regimen for prediction of modifications, we found strongly variegated success rates for the prediction of methylated purines, as exemplified with N1-methyladenosine (m1A). Among the 13 enzymes, a correlation was found between read length, misincorporation, and prediction success. Inversely, low average read length was correlated to high arrest rate and lower prediction success. The three most successful polymerases were then applied to the characterization of RT-signatures of other methylated purines. Guanosines featuring methyl groups on the Watson-Crick face were identified with high confidence, but discrimination between m1G and m22G was only partially successful. In summary, the results suggest that, given sufficient coverage and a set of specifically optimized reaction conditions for reverse transcription, all RNA modifications that impede Watson-Crick bonds can be distinguished by their RT-signature.


Assuntos
DNA Polimerase Dirigida por RNA/metabolismo , Transcrição Reversa , Adenosina/análogos & derivados , Guanosina/química , Guanosina/metabolismo , Aprendizado de Máquina , Metilação , Oligorribonucleotídeos/química , Transcriptoma
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