RESUMO
A growing number of nucleobase modifications in messenger RNA have been revealed through advances in detection and RNA sequencing. Although some of the biochemical pathways that involve modified bases have been identified, research into the world of RNA modification - the epitranscriptome - is still in an early phase. A variety of chemical tools are being used to characterize base modifications, and the structural effects of known base modifications on RNA pairing, thermodynamics and folding are being determined in relation to their putative biological roles.
Assuntos
RNA Mensageiro/análise , RNA Mensageiro/química , Animais , Humanos , Espectrometria de Massas , Estrutura Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de RNA , Transcriptoma/genéticaRESUMO
Direct measurement of DNA repair enzyme activities is important both for the basic study of cellular repair pathways as well as for potential new translational applications in their associated diseases. NTH1, a major glycosylase targeting oxidized pyrimidines, prevents mutations arising from this damage, and the regulation of NTH1 activity is important in resisting oxidative stress and in suppressing tumor formation. Herein, we describe a novel molecular strategy for the direct detection of damaged DNA base excision activity by a ratiometric fluorescence change. This strategy utilizes glycosylase-induced excimer formation of pyrenes, and modified DNA probes, incorporating two pyrene deoxynucleotides and a damaged base, enable the direct, real-time detection of NTH1 activity inâ vitro and in cellular lysates. The probe design was also applied in screening for potential NTH1 inhibitors, leading to the identification of a new small-molecule inhibitor with sub-micromolar potency.
Assuntos
Reparo do DNA , Desoxirribonuclease (Dímero de Pirimidina)/química , Antimutagênicos/farmacologia , Dano ao DNA , Desoxirribonuclease (Dímero de Pirimidina)/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Humanos , Oxirredução , Estresse Oxidativo , Pirimidinas/químicaRESUMO
External photocontrol over RNA function has emerged as a useful tool for studying nucleic acid biology. Most current methods rely on fully synthetic nucleic acids with photocaged nucleobases, limiting application to relatively short synthetic RNAs. Here we report a method to gain photocontrol over RNA by postsynthetic acylation of 2'-hydroxyls with photoprotecting groups. One-step introduction of these groups efficiently blocks hybridization, which is restored after light exposure. Polyacylation (termed cloaking) enables control over a hammerhead ribozyme, illustrating optical control of RNA catalytic function. Use of the new approach on a transcribed 237 nt RNA aptamer demonstrates the utility of this method to switch on RNA folding in a cellular context, and underlines the potential for application in biological studies.
Assuntos
Processos Fotoquímicos , RNA/metabolismo , Acilação , Estrutura Molecular , RNA/químicaRESUMO
The activity of DNA repair enzyme 8-oxoguanine DNA glycosylase (OGG1), which excises oxidized base 8-oxoguanine (8-OG) from DNA, is closely linked to mutagenesis, genotoxicity, cancer, and inflammation. To test the roles of OGG1-mediated repair in these pathways, we have undertaken the development of noncovalent small-molecule inhibitors of the enzyme. Screening of a PubChem-annotated library using a recently developed fluorogenic 8-OG excision assay resulted in multiple validated hit structures, including selected lead hit tetrahydroquinoline 1 (IC50 = 1.7 µM). Optimization of the tetrahydroquinoline scaffold over five regions of the structure ultimately yielded amidobiphenyl compound 41 (SU0268; IC50 = 0.059 µM). SU0268 was confirmed by surface plasmon resonance studies to bind the enzyme both in the absence and in the presence of DNA. The compound SU0268 was shown to be selective for inhibiting OGG1 over multiple repair enzymes, including other base excision repair enzymes, and displayed no toxicity in two human cell lines at 10 µM. Finally, experiments confirm the ability of SU0268 to inhibit OGG1 in HeLa cells, resulting in an increase in accumulation of 8-OG in DNA. The results suggest the compound SU0268 as a potentially useful tool in studies of the role of OGG1 in multiple disease-related pathways.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , DNA Glicosilases/antagonistas & inibidores , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Células CACO-2 , DNA Glicosilases/metabolismo , Guanina/análogos & derivados , Guanina/metabolismo , Células HEK293 , Células HeLa , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/metabolismoRESUMO
Nucleotide surveillance enzymes play important roles in human health, by monitoring damaged monomers in the nucleotide pool and deactivating them before they are incorporated into chromosomal DNA or disrupt nucleotide metabolism. In particular, deamination of cytosine, leading to uracil in DNA and in the nucleotide pool, can be deleterious, causing DNA damage. The enzyme deoxyuridine triphosphatase (dUTPase) is currently under study as a therapeutic and prognostic target for cancer. Measuring the activity of this enzyme is important both in basic research and in clinical applications involving this pathway, but current methods are nonselective, detecting pyrophosphate, which is produced by many enzymes. Here we describe the design and synthesis of a dUTPase enzyme-specific chimeric dinucleotide (DUAL) that replaces the pyrophosphate leaving group of the native substrate with ATP, enabling sensitive detection via luciferase luminescence signaling. The DUAL probe functions sensitively and selectively to quantify enzyme activities in vitro and in cell lysates. We further report the first measurements of dUTPase activities in eight different cell lines, which are found to vary by a factor of 7-fold. We expect that the new probe can be of considerable utility in research involving this clinically significant enzyme.
Assuntos
Trifosfato de Adenosina/análogos & derivados , Substâncias Luminescentes/química , Nucleotídeos/química , Pirofosfatases/análise , Uridina Trifosfato/análogos & derivados , Linhagem Celular Tumoral , Ensaios Enzimáticos/métodos , Células HEK293 , Humanos , Medições Luminescentes/métodos , Especificidade por SubstratoRESUMO
We describe a selective and mild chemical approach for controlling RNA hybridization, folding, and enzyme interactions. Reaction of RNAs in aqueous buffer with an azide-substituted acylating agent (100-200â mm) yields several 2'-OH acylations per RNA strand in as little as 10â min. This poly-acylated ("cloaked") RNA is strongly blocked from hybridization with complementary nucleic acids, from cleavage by RNA-processing enzymes, and from folding into active aptamer structures. Importantly, treatment with a water-soluble phosphine triggers a Staudinger reduction of the azide groups, resulting in spontaneous loss of acyl groups ("uncloaking"). This fully restores RNA folding and biochemical activity.
Assuntos
Azidas/farmacologia , RNA/efeitos dos fármacos , Acilação/efeitos dos fármacos , Azidas/antagonistas & inibidores , Azidas/química , Estrutura Molecular , Fosfinas/química , Fosfinas/farmacologia , Dobramento de RNA/efeitos dos fármacosRESUMO
Posttranscriptional modifications of RNA bases are not only found in many noncoding RNAs but have also recently been identified in coding (messenger) RNAs as well. They require complex and laborious methods to locate, and many still lack methods for localized detection. Here we test the ability of next-generation sequencing (NGS) to detect and distinguish between ten modified bases in synthetic RNAs. We compare ultradeep sequencing patterns of modified bases, including miscoding, insertions and deletions (indels), and truncations, to unmodified bases in the same contexts. The data show widely varied responses to modification, ranging from no response, to high levels of mutations, insertions, deletions, and truncations. The patterns are distinct for several of the modifications, and suggest the future use of ultradeep sequencing as a fingerprinting strategy for locating and identifying modifications in cellular RNAs.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Mutação , RNA/genética , Mutação INDEL , Modelos Moleculares , RNA Mensageiro/genéticaRESUMO
Nanometer-sized fragments of carbon in the form of multilayer graphene ("carbon dots") have been under highly active study for applications in imaging. While offering advantages of low toxicity and photostability, such nanomaterials are inhomogeneous and have limited wavelengths of emission. Here we address these issues by assembling luminescent aromatic C16-C38 hydrocarbons together on a DNA scaffold in homogeneous, soluble molecular compounds. Monomer deoxyribosides of five different aromatic hydrocarbons were synthesized and assembled into a library of 1296 different tetramer compounds on PEG-polystyrene beads. These were screened for photostability and a range of emission colors using 365 nm excitation, observing visible light (>400 nm) emission. We identified a set of six oligomers (DNA-carbon assemblies, DNA-CAs) with exceptional photostability that emit from 400 to 680 nm in water, with Stokes shifts of up to 110 nm, quantum yields ranging from 0.01 to 0.29, and fluorescence lifetimes from 3 to 42 ns. In addition, several of these DNA-CAs exhibited white emission in aqueous solution. The molecules were used in multispectral cell imaging experiments and were taken up into cells passively. The results expand the range of emission properties that can be achieved in water with all-hydrocarbon chromophores and establish the use of the DNA scaffold to arrange carbon layers in homogeneous, rapidly synthesized assemblies.
Assuntos
Carbono/química , DNA/química , Luminescência , Células HeLa , Humanos , Estrutura MolecularRESUMO
The off-target toxicity of drugs targeted to proteins imparts substantial health and economic costs. Proteome interaction studies can reveal off-target effects with unintended proteins; however, little attention has been paid to intracellular RNAs as potential off-targets that may contribute to toxicity. To begin to assess this, we developed a reactivity-based RNA profiling methodology and applied it to uncover transcriptome interactions of a set of Food and Drug Administration-approved small-molecule drugs in vivo. We show that these protein-targeted drugs pervasively interact with the human transcriptome and can exert unintended biological effects on RNA functions. In addition, we show that many off-target interactions occur at RNA loci associated with protein binding and structural changes, allowing us to generate hypotheses to infer the biological consequences of RNA off-target binding. The results suggest that rigorous characterization of drugs' transcriptome interactions may help assess target specificity and potentially avoid toxicity and clinical failures.
RESUMO
Impaired DNA repair activity has been shown to greatly increase rates of cancer clinically. It has been hypothesized that upregulating repair activity in susceptible individuals may be a useful strategy for inhibiting tumorigenesis. Here, we report that selected tyrosine kinase (TK) inhibitors including nilotinib, employed clinically in the treatment of chronic myeloid leukemia, are activators of the repair enzyme Human MutT Homolog 1 (MTH1). MTH1 cleanses the oxidatively damaged cellular nucleotide pool by hydrolyzing the oxidized nucleotide 8-oxo-2'-deoxyguanosine (8-oxo-dG)TP, which is a highly mutagenic lesion when incorporated into DNA. Structural optimization of analogues of TK inhibitors resulted in compounds such as SU0448, which induces 1000 ± 100% activation of MTH1 at 10 µM and 410 ± 60% at 5 µM. The compounds are found to increase the activity of the endogenous enzyme, and at least one (SU0448) decreases levels of 8-oxo-dG in cellular DNA. The results suggest the possibility of using MTH1 activators to decrease the frequency of mutagenic nucleotides entering DNA, which may be a promising strategy to suppress tumorigenesis in individuals with elevated cancer risks.
Assuntos
Enzimas Reparadoras do DNA/metabolismo , Neoplasias , Monoéster Fosfórico Hidrolases/metabolismo , 8-Hidroxi-2'-Desoxiguanosina , Carcinogênese , DNA , Dano ao DNA , Humanos , Nucleotídeos , Estresse OxidativoRESUMO
We describe the synthesis and RNA acylation activity of a series of minimalist azidoalkanoyl imidazole reagents, with the aim of functionalizing RNA at 2'-hydroxyl groups at stoichiometric to superstoichiometric levels. We find marked effects of small structural changes on their ability to acylate and be reductively removed, and identify reagents and methods that enable efficient RNA functionalization and control.
Assuntos
Azidas/química , Imidazóis/química , Indicadores e Reagentes/química , RNA/química , Acilação , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Azidas/síntese química , Imidazóis/síntese química , Indicadores e Reagentes/síntese química , Estrutura Molecular , Hibridização de Ácido Nucleico , Oxirredução , Fosfinas/química , RNA/genética , Dobramento de RNARESUMO
Acylation of RNA at 2'-OH groups is widely applied in mapping RNA structure and recently for controlling RNA function. Reactions are described that install the smallest 2-carbon acyl groups on RNA-namely, 2'-O-acetyl and 2'-O-carbonate groups. Hybridization and thermal melting experiments are performed to assess the effects of the acyl groups on duplex formation. Both reagents can be employed at lower concentrations to map RNA secondary structure by reverse transcriptase primer extension (SHAPE) methods.
Assuntos
Acetatos/química , Cimento de Policarboxilato/química , RNA/química , Acilação , Sequência de Bases , Indicadores e Reagentes/química , RNA/genéticaRESUMO
Oxidative damage in DNA is one of the primary sources of mutations in the cell. The activities of repair enzymes 8-oxoguanine DNA glycosylase (OGG1) and human MutT Homologue 1 (NUDT1 or MTH1), which work together to ameliorate this damage, are closely linked to mutagenesis, genotoxicity, cancer, and inflammation. Here we have undertaken the development of small-molecule dual inhibitors of the two enzymes as tools to test the relationships between these pathways and disease. The compounds preserve key structural elements of known inhibitors of the two enzymes, and they were synthesized and assayed with recently developed luminescence assays of the enzymes. Further structural refinement of initial lead molecules yielded compound 5 (SU0383) with IC50(NUDT1) = 0.034 µM and IC50(OGG1) = 0.49 µM. The compound SU0383 displayed low toxicity in two human cell lines at 10 µM. Experiments confirm the ability of SU0383 to increase sensitivity of tumor cells to oxidative stress. Dual inhibitors of these two enzymes are expected to be useful in testing multiple hypotheses regarding the roles of 8-oxo-dG in multiple disease states.
Assuntos
DNA Glicosilases/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Guanina/análogos & derivados , Monoéster Fosfórico Hidrolases/metabolismo , Animais , Linhagem Celular Tumoral , DNA Glicosilases/antagonistas & inibidores , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Guanina/antagonistas & inibidores , HumanosRESUMO
We report the development of post-transcriptional chemical methods that enable control over CRISPR-Cas9 gene editing activity both in in vitro assays and in living cells. We show that an azide-substituted acyl imidazole reagent (NAI-N3) efficiently acylates CRISPR single guide RNAs (sgRNAs) in 20 minutes in buffer. Poly-acylated ("cloaked") sgRNA was completely inactive in DNA cleavage with Cas9 in vitro, and activity was quantitatively restored after phosphine treatment. Delivery of cloaked sgRNA and Cas9 mRNA into HeLa cells was enabled by the use of charge-altering releasable transporters (CARTs), which outperformed commercial transfection reagents in transfecting sgRNA co-complexed with Cas9 encoding functional mRNA. Genomic DNA cleavage in the cells by CRISPR-Cas9 was efficiently restored after treatment with phosphine to remove the blocking acyl groups. Our results highlight the utility of reversible RNA acylation as a novel method for temporal control of genome-editing function.
RESUMO
Hydrazone and oxime bond formation between α-nucleophiles (e.g. hydrazines, alkoxy-amines) and carbonyl compounds (aldehydes and ketones) is convenient and is widely applied in multiple fields of research. While the reactants are simple, a substantial drawback is the relatively slow reaction at neutral pH. Here we describe a novel molecular strategy for accelerating these reactions, using bifunctional buffer compounds that not only control pH but also catalyze the reaction. The buffers can be employed at pH 5-9 (5-50 mM) and accelerate reactions by several orders of magnitude, yielding second-order rate constants of >10 M-1 s-1. Effective bifunctional amines include 2-(aminomethyl)imidazoles and N,N-dimethylethylenediamine. Unlike previous diaminobenzene catalysts, the new buffer amines are found to have low toxicity to human cells, and can be used to promote reactions in cellular applications.
RESUMO
Human ribonuclease Dicer is an enzyme that excises small regulatory RNAs from perfectly or partially double-stranded RNA precursors. Although Dicer substrates and products have already been quite well characterized, our knowledge about cellular factors regulating the activity of this enzyme is still limited. To learn more about this problem, we attempted to determine whether RNA could function not only as a Dicer substrate but also as its regulator. To this end, we applied an in vitro selection method. We identified 120 RNA oligomers binding human Dicer. Sixteen of them were subjected to more detailed in vitro studies. We found that 6 out of 16 oligomers affected Dicer ability to digest pre-microRNAs (miRNAs), although most of them were cleaved by this enzyme. For the 6 most active oligomers the putative mechanism of Dicer inhibition was determined. Three oligomers were classified as typical competitive inhibitors and one as an allosteric inhibitor. The remaining 2 oligomers acted as selective inhibitors. They affected the production of 1 miRNA, whereas the formation of other miRNAs was hardly influenced. In general, the data obtained suggest that one can modulate the generation of specific miRNAs by using RNA oligomers. Moreover, we found that sequences similar to those of the selected oligomers can be found within the molecules composing human transcriptome.
Assuntos
Aptâmeros de Nucleotídeos/química , RNA Helicases DEAD-box/antagonistas & inibidores , Ribonuclease III/antagonistas & inibidores , Regulação Alostérica , Aptâmeros de Nucleotídeos/farmacologia , Sequência de Bases , Ligação Competitiva , Simulação por Computador , Ensaios Enzimáticos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , MicroRNAs/genética , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Técnica de Seleção de Aptâmeros , TranscriptomaRESUMO
A ribosome undergoes significant conformational changes during elongation of polypeptide chain that are correlated with structural changes of rRNAs. We tested nine different antisense oligodeoxynucleotides complementary to the selected, highly conserved sequences of Lupinus luteus 26S rRNA that are engaged in the interactions with tRNA molecules. The ribosomes were converted either to pre- or to posttranslocational states, with or without prehybridized oligonucleotides, using tRNA or mini-tRNA molecules. The activity of those ribosomes was tested via the so-called binding assay. We observed well-defined structural changes of ribosome's conformation during different steps of the elongation cycle of protein biosynthesis. In this article, we present that (i) before and after translocation, fragments of domain V between helices H70/H71 and H74/H89 do not have to interact with nucleotides 72-76 of the acceptor arm of A-site tRNA; (ii) helix H69 does not have to interact with DHU arm of tRNA in positions 25 and 26 after forming the peptide bond, but before translocation; (iii) helices H69 and H70 interact weakly with nucleotides 11, 12, 25, and 26 of A-site tRNA before forming a peptide bond in the ribosome; (iv) interactions between helices H80, H93 and single-stranded region between helices H92 and H93 and CCAend of P-site tRNA are necessary at all steps of elongation cycle; and (v) before and after translocation, helix H89 does not have to interact with nucleotides in positions 64-65 and 50-53 of A-site tRNA TPsiC arm.