RESUMO
The methyltransferase enzymes can be applied to deliver a range of modifications to pre-determined sites on large DNA molecules with exceptional specificity and efficiency. To date, however, a limited number of modifications have been delivered in this way because of the complex chemical synthesis that is needed to produce a cofactor analogue carrying a specific function, such as a fluorophore. Here, we describe a method for the direct transfer of a series of functional compounds (seven fluorescent dyes, biotin and polyethylene glycol) to the DNA duplex. Our approach uses a functional cofactor analogue, whose final preparative step is performed alongiside the DNA modification reaction in a single pot, with no purification needed. We show that fluorophore conjugation efficiency in these mixtures is significantly improved compared to two-step labeling approaches. Our experiments highlight the remarkable malleability and selectivity of the methyltransferases tested. Additional analysis using high resolution localization of the fluorophore distribution indicates that target sites for the methyltransferase are predominantly labeled on a single strand of their palindromic site and that a small and randomly-distributed probability of off-site labeling exists.
Assuntos
Biotina/química , DNA/química , Corantes Fluorescentes/química , Metiltransferases/metabolismo , Polietilenoglicóis/química , Alquilação , Biocatálise , Plasmídeos/genéticaRESUMO
Methyltransferases (MTases) form a large family of enzymes that methylate a diverse set of targets, ranging from the three major biopolymers to small molecules. Most of these MTases use the cofactor S-adenosyl-l-Methionine (AdoMet) as a methyl source. In recent years, there have been significant efforts toward the development of AdoMet analogues with the aim of transferring moieties other than simple methyl groups. Two major classes of AdoMet analogues currently exist: doubly-activated molecules and aziridine based molecules, each of which employs a different approach to achieve transalkylation rather than transmethylation. In this review, we discuss the various strategies for labelling and functionalizing biomolecules using AdoMet-dependent MTases and AdoMet analogues. We cover the synthetic routes to AdoMet analogues, their stability in biological environments and their application in transalkylation reactions. Finally, some perspectives are presented for the potential use of AdoMet analogues in biology research, (epi)genetics and nanotechnology.
Assuntos
Biopolímeros/metabolismo , Metiltransferases/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismo , Biopolímeros/química , Metiltransferases/química , Bibliotecas de Moléculas Pequenas/químicaRESUMO
We demonstrate an approach to optical DNA mapping, which enables near single-molecule characterization of whole bacteriophage genomes. Our approach uses a DNA methyltransferase enzyme to target labelling to specific sites and copper-catalysed azide-alkyne cycloaddition to couple a fluorophore to the DNA. We achieve a labelling efficiency of â¼70% with an average labelling density approaching one site every 500 bp. Such labelling density bridges the gap between the output of a typical DNA sequencing experiment and the long-range information derived from traditional optical DNA mapping. We lay the foundations for a wider-scale adoption of DNA mapping by screening 11 methyltransferases for their ability to direct sequence-specific DNA transalkylation; the first step of the DNA labelling process and by optimizing reaction conditions for fluorophore coupling via a click reaction. Three of 11 enzymes transalkylate DNA with the cofactor we tested (a readily prepared s-adenosyl-l-methionine analogue).
Assuntos
Química Click , Metilases de Modificação do DNA/metabolismo , DNA/química , Genômica/métodos , Alquilação , DNA/metabolismo , Dano ao DNA , Corantes Fluorescentes , S-Adenosilmetionina/análogos & derivados , S-Adenosilmetionina/químicaRESUMO
Single-molecule DNA mapping has the potential to serve as a powerful complement to high-throughput sequencing in metagenomic analysis. Offering longer read lengths and forgoing the need for complex library preparation and amplification, mapping stands to provide an unbiased view into the composition of complex viromes and/or microbiomes. To fully enable mapping-based metagenomics, sensitivity and specificity of DNA map analysis and identification need to be improved. Using detailed simulations and experimental data, we first demonstrate how fluorescence imaging of surface stretched, sequence specifically labeled DNA fragments can yield highly sensitive identification of targets. Second, a new analysis technique is introduced to increase specificity of the analysis, allowing even closely related species to be resolved. Third, we show how an increase in resolution improves sensitivity. Finally, we demonstrate that these methods are capable of identifying species with long genomes such as bacteria with high sensitivity.
RESUMO
We report an assay for determining the number of fluorophores conjugated to single plasmid DNA molecules and apply this to compare the efficiency of fluorophore coupling strategies for covalent DNA labelling. We compare a copper-catalyzed azide-alkyne cycloaddition reaction, amine to N-hydroxysuccinimidyl ester coupling reaction and strain-promoted azide-alkyne cycloaddition reaction for fluorescent DNA labelling. We found increased labelling efficiency going from the amine to N-hydroxysuccinimidyl ester coupling reaction to the copper-catalyzed azide-alkyne cycloaddition and found the highest degree of DNA labelling with the strain-promoted azide-alkyne cycloaddition reaction. We also examined the effect of labelling on the DNA structure using atomic force microscopy. We observe no distortions or damage to the DNA that was labeled using the amine to N-hydroxysuccinimidyl ester and strain-promoted azide-alkyne cycloaddition coupling reactions. This was in contrast to the copper-catalyzed azide-alkyne cycloaddition reaction, which, despite the use of copper-coordinating ligands in the labelling mixture, leads to some structural DNA damage (single-stranded DNA breaks).
RESUMO
Deposition of linear DNA molecules is a critical step in many single-molecule genomic approaches including DNA mapping, fiber-FISH, and several emerging sequencing technologies. In the ideal situation, the DNA that is deposited for these experiments is absolutely linear and uniformly stretched, thereby enabling accurate distance measurements. However, this is rarely the case, and furthermore, current approaches for the capture and linearization of DNA on a surface tend to require complex surface preparation and large amounts of starting material to achieve genomic-scale mapping. This makes them technically demanding and prevents their application in emerging fields of genomics, such as single-cell based analyses. Here we describe a simple and extremely efficient approach to the deposition and linearization of genomic DNA molecules. We employ droplets containing as little as tens of picograms of material and simply drag them, using a pipet tip, over a polymer-coated coverslip. In this report we highlight one particular polymer, Zeonex, which is remarkably efficient at capturing DNA. We characterize the method of DNA capture on the Zeonex surface and find that the use of droplets greatly facilitates the efficient deposition of DNA. This is the result of a circulating flow in the droplet that maintains a high DNA concentration at the interface of the surface/solution. Overall, our approach provides an accessible route to the study of genomic structural variation from samples containing no more than a handful of cells.
Assuntos
Genoma Humano , Ácidos Nucleicos Imobilizados/química , Fenômenos Mecânicos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Conformação de Ácido Nucleico , Polímeros/química , Propriedades de SuperfícieRESUMO
The technologies associated with DNA sequencing are rapidly evolving. Indeed, single-molecule DNA sequencing strategies are cheaper and faster than ever before. Despite this progress, every sequencing platform to date relies on reading the genome in small, abstract fragments, typically of less than 1000 bases in length. The overarching aim of the optical map is to complement the information derived from DNA sequencing by providing long-range context on which these short sequence reads can be built. This is typically done using an enzyme to target and modify at short DNA sequences of, say, six bases in length throughout the genome. By accurately placing these short pieces of sequence on long genomic DNA fragments, up to several millions of bases in length, a scaffold for sequence assembly can be obtained. This review focuses on three enzymatic approaches to optical mapping. Optical mapping was first developed using restriction enzymes to sequence-specifically cleave DNA that is immobilized on a surface. More recently, nicking enzymes have found application in the sequence-specific fluorescent labeling of DNA for optical mapping. Such covalent modification allows the DNA to be imaged in solution, and this, in combination with developing nanofluidic technologies, is enabling new high-throughput approaches to mapping. And, finally, this review will discuss the recent development of mapping with subdiffraction-limit precision using methyltransferase enzymes to label the DNA with an ultrahigh density.