Robert K. Neely

Localizer: fast, accurate, open-source, and modular software package for superresolution microscopy

Abstract. We present Localizer, a freely available and open source software package that implements the computational data processing inherent to several types of superresolution fluorescence imaging, such as localization (PALM/STORM/GSDIM) and fluctuation imaging (SOFI/pcSOFI). Localizer delivers high accuracy and performance and comes with a fully featured and easy-to-use graphical user interface but is also designed to be integrated in higher-level analysis environments. Due to its modular design, Localizer can be readily extended with new algorithms as they become available, while maintaining the same interface and performance. We provide front-ends for running Localizer from Igor Pro, Matlab, or as a stand-alone program. We show that Localizer performs favorably when compared with two existing superresolution packages, and to our knowledge is the only freely available implementation of SOFI/pcSOFI microscopy. By dramatically improving the analysis performance and ensuring the easy addition of current and future enhancements, Localizer strongly improves the usability of superresolution imaging in a variety of biomedical studies.

Optical Mapping of DNA: Single-Molecule-Based Methods for Mapping Genomes

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.

Time-resolved fluorescence of 2-aminopurine as a probe of base flipping in M.HhaI–DNA complexes

DNA base flipping is an important mechanism in molecular enzymology, but its study is limited by the lack of an accessible and reliable diagnostic technique. A series of crystalline complexes of a DNA methyltransferase, M.HhaI, and its cognate DNA, in which a fluorescent nucleobase analogue, 2-aminopurine (AP), occupies defined positions with respect the target flipped base, have been prepared and their structures determined at higher than 2 Å resolution. From time-resolved fluorescence measurements of these single crystals, we have established that the fluorescence decay function of AP shows a pronounced, characteristic response to base flipping: the loss of the very short (∼100 ps) decay component and the large increase in the amplitude of the long (∼10 ns) component. When AP is positioned at sites other than the target site, this response is not seen. Most significantly, we have shown that the same clear response is apparent when M.HhaI complexes with DNA in solution, giving an unambiguous signal of base flipping. Analysis of the AP fluorescence decay function reveals conformational heterogeneity in the DNA–enzyme complexes that cannot be discerned from the present X-ray structures.

DNA fluorocode: A single molecule, optical map of DNA with nanometre resolution

We present a new method for single-molecule optical DNA mapping using an exceptionally dense, yet sequence-specific coverage of DNA with a fluorescent probe. The method employs a DNA methyltransferase enzyme to direct the DNA labelling, followed by molecular combing of the DNA onto a polymer-coated surface and subsequent sub-diffraction limit localization of the fluorophores. The result is a ‘DNA fluorocode’; a simple description of the DNA sequence, with a maximum achievable resolution of less than 20 bases, which can be read and analyzed like a barcode. We demonstrate the generation of a fluorocode for genomic DNA from the lambda bacteriophage using a DNA methyltransferase, M.HhaI, to direct fluorescent labels to four-base sequences reading 5′-GCGC-3′. A consensus fluorocode that allows the study of the DNA sequence at the level of an individual labelling site can be generated from a handful of molecules.

Super-resolution optical DNA Mapping via DNA methyltransferase-directed click chemistry

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 ∼70% 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).

2-aminopurine as a fluorescent probe of DNA conformation and the DNA–enzyme interface

Nearly 50 years since its potential as a fluorescent base analogue was first recognized, 2-aminopurine (2AP) continues to be the most widely used fluorescent probe of DNA structure and the perturbation of that structure by interaction with enzymes and other molecules. In this review, we begin by considering the origin of the dramatic and intriguing difference in photophysical properties between 2AP and its structural isomer, adenine; although 2AP differs from the natural base only in the position of the exocyclic amine group, its fluorescence intensity is one thousand times greater. We then discuss the mechanism of interbase quenching of 2AP fluorescence in DNA, which is the basis of its use as a conformational probe but remains imperfectly understood. There are hundreds of examples in the literature of the use of changes in the fluorescence intensity of 2AP as the basis of assays of conformational change; however, in this review we will consider in detail only a few intensity-based studies. Our primary aim is to highlight the use of time-resolved fluorescence measurements, and the interpretation of fluorescence decay parameters, to explore the structure and dynamics of DNA. We discuss the salient features of the fluorescence decay of 2AP when incorporated in DNA and review the use of decay measurements in studying duplexes, single strands and other structures. We survey the use of 2AP as a probe of DNA-enzyme interaction and enzyme-induced distortion, focusing particularly on its use to study base flipping and the enhanced mechanistic insights that can be gained by a detailed analysis of the decay parameters, rather than merely monitoring changes in fluorescence intensity. Finally we reflect on the merits and shortcomings of 2AP and the prospects for its wider adoption as a fluorescence-decay-based probe.

Time-resolved fluorescence studies of nucleotide flipping by restriction enzymes

Restriction enzymes Ecl18kI, PspGI and EcoRII-C, specific for interrupted 5-bp target sequences, flip the central base pair of these sequences into their protein pockets to facilitate sequence recognition and adjust the DNA cleavage pattern. We have used time-resolved fluorescence spectroscopy of 2-aminopurine-labelled DNA in complex with each of these enzymes in solution to explore the nucleotide flipping mechanism and to obtain a detailed picture of the molecular environment of the extrahelical bases. We also report the first study of the 7-bp cutter, PfoI, whose recognition sequence (T/CCNGGA) overlaps with that of the Ecl18kI-type enzymes, and for which the crystal structure is unknown. The time-resolved fluorescence experiments reveal that PfoI also uses base flipping as part of its DNA recognition mechanism and that the extrahelical bases are captured by PfoI in binding pockets whose structures are quite different to those of the structurally characterized enzymes Ecl18kI, PspGI and EcoRII-C. The fluorescence decay parameters of all the enzyme-DNA complexes are interpreted to provide insight into the mechanisms used by these four restriction enzymes to flip and recognize bases and the relationship between nucleotide flipping and DNA cleavage.

Combing of Genomic DNA from Droplets Containing Picograms of Material

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.

De novo Identification of DNA Modifications Enabled by Genome-Guided Nanopore Signal Processing

Advances in single molecule sequencing technology have enabled the investigation of the full catalogue of covalent DNA modifications. We present an assay, Modified DNA sequencing (MoD-seq), that leverages raw nanopore data processing, visualization and statistical testing to directly survey DNA modifications without the need for a large prior training dataset. We present case studies applying MoD-seq to identify three distinct marks, 4mC, 5mC, and 6mA, and demonstrate quantitative reproducibility across biological replicates processed in different labs. In a ground-truth dataset created via in vitro treatment of synthetic DNA with selected methylases, we show that modifications can be detected in a variety of distinct sequence contexts. We recapitulated known methylation patterns and frequencies in E. coli, and propose a pipeline for the comprehensive discovery of DNA modifications in a genome without a priori knowledge of their chemical identities.

Use of time‐resolved FRET to validate crystal structure of complement regulatory complex between C3b and factor H (N terminus)

Structural knowledge of interactions amongst the ∼ 40 proteins of the human complement system, which is central to immune surveillance and homeostasis, is expanding due primarily to X‐ray diffraction of co‐crystallized proteins. Orthogonal evidence, in solution, for the physiological relevance of such co‐crystal structures is valuable since intermolecular affinities are generally weak‐to‐medium and inter‐domain mobility may be important. In this current work, Förster resonance energy transfer (FRET) was used to investigate the 10 μM KD (210 kD) complex between the N‐terminal region of the soluble complement regulator, factor H (FH1‐4), and the key activation‐specific complement fragment, C3b. Using site‐directed mutagenesis, seven cysteines were introduced individually at potentially informative positions within the four CCP modules comprising FH1‐4, then used for fluorophore attachment. C3b possesses a thioester domain featuring an internal cycloglutamyl cysteine thioester; upon hydrolysis this yields a free thiol (Cys988) that was also fluorescently tagged. Labeled proteins were functionally active as cofactors for cleavage of C3b to iC3b except for FH1‐4(Q40C) where conjugation with the fluorophore likely abrogated interaction with the protease, factor I. Time‐resolved FRET measurements were undertaken to explore interactions between FH1‐4 and C3b in fluid phase and under near‐physiological conditions. These experiments confirmed that, as in the cocrystal structure, FH1‐4 binds to C3b with CCP module 1 furthest from, and CCP module 4 closest to, the thioester domain, placing subsequent modules of FH near to any surface to which C3b is attached. The data do not rule out flexibility of the thioester domain relative to the remainder of the complex.