Victoria Birkedal

Self-assembly of a nanoscale DNA box with a controllable lid

The unique structural motifs and self-recognition properties of DNA can be exploited to generate self-assembling DNA nanostructures of specific shapes using a ‘bottom-up’ approach. Several assembly strategies have been developed for building complex three-dimensional (3D) DNA nanostructures. Recently, the DNA ‘origami’ method was used to build two-dimensional addressable DNA structures of arbitrary shape that can be used as platforms to arrange nanomaterials with high precision and specificity. A long-term goal of this field has been to construct fully addressable 3D DNA nanostructures. Here we extend the DNA origami method into three dimensions by creating an addressable DNA box 42 × 36 × 36 nm3 in size that can be opened in the presence of externally supplied DNA ‘keys’. We thoroughly characterize the structure of this DNA box using cryogenic transmission electron microscopy, small-angle X-ray scattering and atomic force microscopy, and use fluorescence resonance energy transfer to optically monitor the opening of the lid. Controlled access to the interior compartment of this DNA nanocontainer could yield several interesting applications, for example as a logic sensor for multiple-sequence signals or for the controlled release of nanocargos.

Construction of a 4 Zeptoliters Switchable 3D DNA Box Origami

The DNA origami technique is a recently developed self-assembly method that allows construction of 3D objects at the nanoscale for various applications. In the current study we report the production of a 18 × 18 × 24 nm(3) hollow DNA box origami structure with a switchable lid. The structure was efficiently produced and characterized by atomic force microscopy, transmission electron microscopy, and Förster resonance energy transfer spectroscopy. The DNA box has a unique reclosing mechanism, which enables it to repeatedly open and close in response to a unique set of DNA keys. This DNA device can potentially be used for a broad range of applications such as controlling the function of single molecules, controlled drug delivery, and molecular computing.

Tunable light source for coherent anti-Stokes Raman scattering microspectroscopy based on the soliton self-frequency shift

We present a photonic crystal fiber (PCF)-based light source for generating tunable excitation pulses (pump and Stokes) that are applicable to coherent anti-Stokes Raman scattering (CARS) microspectroscopy. The laser employed is an unamplified Ti:sapphire femtosecond laser oscillator. The CARS pump pulse is generated by spectral compression of a laser pulse in a PCF. The Stokes pulse is generated by redshifting a laser pulse in a PCF through the soliton self-frequency shift. This setup allows for probing up to 4000 cm(-1) with a spectral resolution of approximately 25 cm(-1). We characterize the stability and robustness of CARS microspectroscopy employing this light source.

Routing of individual polymers in designed patterns

Synthetic polymers are ubiquitous in the modern world, but our ability to exert control over the molecular conformation of individual polymers is very limited. In particular, although the programmable self-assembly of oligonucleotides and proteins into artificial nanostructures has been demonstrated, we currently lack the tools to handle other types of synthetic polymers individually and thus the ability to utilize and study their single-molecule properties. Here we show that synthetic polymer wires containing short oligonucleotides that extend from each repeat can be made to assemble into arbitrary routings. The wires, which can be more than 200 nm in length, are soft and bendable, and the DNA strands allow individual polymers to self-assemble into predesigned routings on both two- and three-dimensional DNA origami templates. The polymers are conjugated and potentially conducting, and could therefore be used to create molecular-scale electronic or optical wires in arbitrary geometries.

Temperature-controlled encapsulation and release of an active enzyme in the cavity of a self-assembled DNA nanocage

We demonstrate temperature-controlled encapsulation and release of the enzyme horseradish peroxidase using a preassembled and covalently closed three-dimensional DNA cage structure as a controllable encapsulation device. The utilized cage structure was covalently closed and composed of 12 double-stranded B-DNA helices that constituted the edges of the structure. The double stranded helices were interrupted by short single-stranded thymidine linkers constituting the cage corners except for one, which was composed by four 32 nucleotide long stretches of DNA with a sequence that allowed them to fold into hairpin structures. As demonstrated by gel-electrophoretic and fluorophore-quenching experiments this design imposed a temperature-controlled conformational transition capability to the structure, which allowed entrance or release of an enzyme cargo at 37 °C while ensuring retainment of the cargo in the central cavity of the cage at 4 °C. The entrapped enzyme was catalytically active inside the DNA cage and was able to convert substrate molecules penetrating the apertures in the DNA lattice that surrounded the central cavity of the cage.

Construction of a fuzzy and Boolean logic gates based on DNA.

Logic gates are devices that can perform logical operations by transforming a set of inputs into a predictable single detectable output. The hybridization properties, structure, and function of nucleic acids can be used to make DNA-based logic gates. These devices are important modules in molecular computing and biosensing. The ideal logic gate system should provide a wide selection of logical operations, and be integrable in multiple copies into more complex structures. Here we show the successful construction of a small DNA-based logic gate complex that produces fluorescent outputs corresponding to the operation of the six Boolean logic gates AND, NAND, OR, NOR, XOR, and XNOR. The logic gate complex is shown to work also when implemented in a three-dimensional DNA origami box structure, where it controlled the position of the lid in a closed or open position. Implementation of multiple microRNA sensitive DNA locks on one DNA origami box structure enabled fuzzy logical operation that allows biosensing of complex molecular signals. Integrating logic gates with DNA origami systems opens a vast avenue to applications in the fields of nanomedicine for diagnostics and therapeutics.

A DNA-Mediated Homogeneous Binding Assay for Proteins and Small Molecules

Optical detection of molecular targets typically requires immobilization, separation, or chemical or enzymatic processing. An important exception is aptamers that allow optical detection in solution based on conformational changes. This method, however, requires the laborious selection of aptamers with high target specificity and affinity, and the ability to undergo the required conformational changes. Here we report on an alternative generic scheme for detecting small molecules and proteins in solution based on a shift in the equilibrium of DNA-based strand displacement competition reaction. The shift occurs upon binding of a protein, for example, an antibody to its target. We demonstrate nanomolar detection of small molecules such as biotin, digoxigenin, vitamin D, and folate, in buffer and in plasma. The method is flexible, and we also show nanomolar detection of the respective antibodies or protein targets of these molecules. The detection scheme provides a generic alternative to aptamers for detection of analytes.

Folding dynamics and conformational heterogeneity of human telomeric G-quadruplex structures in Na+ solutions by single molecule FRET microscopy

G-quadruplex structures can occur throughout the genome, including at telomeres. They are involved in cellular regulation and are potential drug targets. Human telomeric G-quadruplex structures can fold into a number of different conformations and show large conformational diversity. To elucidate the different G-quadruplex conformations and their dynamics, we investigated telomeric G-quadruplex folding using single molecule FRET microscopy in conditions where it was previously believed to yield low structural heterogeneity. We observed four FRET states in Na+ buffers: an unfolded state and three G-quadruplex related states that can interconvert between each other. Several of these states were almost equally populated at low to medium salt concentrations. These observations appear surprising as previous studies reported primarily one G-quadruplex conformation in Na+ buffers. Our results permit, through the analysis of the dynamics of the different observed states, the identification of a more stable G-quadruplex conformation and two transient G-quadruplex states. Importantly these results offer a unique view into G-quadruplex topological heterogeneity and conformational dynamics.

iSMS: single-molecule FRET microscopy software

and conformational dynamics of biomolecules1–3. By reporting on nanoscale distances within single biomolecules, this technique provides the means to uncover population heterogeneity that is normally hidden behind an ensemble mean as well as to monitor the dynamic equilibria of unsynchronized samples4. smFRET additionally holds the potential to determine the structure of dynamic molecules in solution at low molecular concentration5. However, the processing, analysis and interpretation of the large amounts of raw image data in smFRET experiments can be complex and time consuming. Here we present iSMS: an interactive toolkit for the comprehensive analysis of smFRET TIRF-microscopy data (Fig. 1a and Supplementary Software). iSMS integrates and automates common procedures in smFRET data analysis: molecule localization, intensity-trace integration, quantitative FRET determination, FRET distribution analysis, molecule subpopulation analysis and transition state dynamics analysis (Supplementary Notes 1–6 and Supplementary Figs. 1–10). The software provides tools for interacting visually with the data and enables the production of publication-friendly figures. The strengths of iSMS lie in its immediate usability, flexibility and analysis speed and in the several built-in tools for extracting, evaluating and clustering all molecules identified in the raw image data. The entire program can be operated without any prior programming skills and allows interactive sessions of raw and processed data to be easily shared, explored and evaluated by peers. iSMS processes image data almost 20 times faster than current software standards (Supplementary Note 7, Supplementary Table 1 and Supplementary Fig. 11) and is used both in parallel to and after data acquisition. Parallel computing is supported for time-consuming routines, and tools for monitoring and allocating memory are built into the interface. The toolkit can be applied to analyze raw video data from most experimental setups and major microscopy and camera suppliers (Supplementary Note 1). Several robust algorithms help automate the data analysis via interactive tools (Supplementary Notes 2–6). Automation includes emission-channel registration, drift correction, peak localization, FRET-pair identification, intensity integration, FRET iSMS: single-molecule FRET microscopy software

A direct view of the complex multi-pathway folding of telomeric G-quadruplexes

G-quadruplexes (G4s) are DNA secondary structures that are capable of forming and function in vivo. The propensity of G4s to exhibit extreme polymorphism and complex dynamics is likely to influence their cellular function, yet a clear microscopic picture of their folding process is lacking. Here we employed single-molecule FRET microscopy to obtain a direct view of the folding and underlying conformational dynamics of G4s formed by the human telomeric sequence in potassium containing solutions. Our experiments allowed detecting several folded states that are populated in the course of G4 folding and determining their folding energetics and timescales. Combining the single-molecule data with molecular dynamics simulations enabled obtaining a structural description of the experimentally observed folded states. Our work thus provides a comprehensive thermodynamic and kinetic description of the folding of G4s that proceeds through a complex multi-route pathway, involving several marginally stable conformational states.