1. DNA Biosensors

Robert Crawford, Achillefs Kapanidis

DNA is an extremely versatile material with which to build biosensors; molecules can be encoded with binding sites specific for proteins of interest, or for other nucleic acids (DNA/RNA), and can be easily modified to contain useful chemical groups such as fluorophores, biotin or quantum dots. Employing self-assembly properties of DNA, one can also design topological features to create different 2- or 3-dimensional shapes of sizes similar to biomolecular targets. This additional layer of freedom allows even more creativity when designing biosensors. Our lab develops biosensors for the detection of DNA-binding proteins and nucleic acids.

1.1 Detection of DNA-binding proteins
We have developed DNA-based biosensors to detect DNA-binding proteins known as transcription factors (TFs). TFs are proteins that recognise specific sequences on DNA are directly involved in gene regulation. Regulation occurs in response to a number of environmental conditions and signalling molecules (such as amino-acids, nucleotides, heavy metals and hormones) and is achieved through the enhancement or obstruction of RNA polymerase recruitment to promoters. Thus the level and state of TFs can act as a diagnostic tool for cell health or development stage.
Our two published assays [1,2] (see Publications) are based on two different methods for the detection of TFs. The first (Fig 1.), uses two DNA ‘half-sites’, each containing half the binding site for the TF of interest and a 6 nt overhang. Only in the presence of that specific TF are the two half-sites held together, resulting in a change in stoichiometry (available using ALEX; see ‘Methods’) that allows TF detection over a wide concentration range (down to pM levels). Such an assay can be used to detect TFs in cell lysates and also to detect small molecules affecting TF binding.

Figure 1. Half-site based transcription factor (TF) detection. Association of the two DNA fragments in the presence of the specific TF yields an observable change in stoichiometry using ALEX spectroscopy.
Our second assay (Fig 2.) employs the use of FRET (also available using ALEX; see ‘Methods’) for the detection of TFs – in this case Catabolite Activator Protein (CAP). As CAP is well known to significantly deform its DNA site on binding, FRET can be used as the observable for CAP detection. However due to the range of FRET being 2-10 nm, we needed to kink the DNA to bring the ends within this detection range. The use of 5 unpaired adenines creates a ~90˚ kink in the DNA, and three of these kinks was enough to create the working biosensor. This DNA sensor was able to detect CAP on surfaces, in solution, in purified form and expressed in a cell lysate.

Figure 2. FRET-based transcription factor detection (TF). TFs known to bend DNA such as catabolite activator protein (CAP) can be detected using such a design. Three A5 kinks in the DNA bring the ends into a detectible FRET range. TF presence is indicated by bending of one edge.
1.2 Detection of nucleic acids
Nucleic acids specific to particular organisms can be an especially useful biomarker when identifying specific pathogens for example methicillin-resistant Staphylococcus aureus (MRSA) – a significant problem in today’s hospitals. Our lab is currently developing a molecular ‘computational toolkit’ that should allow intelligent sensing of nucleic acids based on simple Boolean logic (AND, OR, NOT). Such a toolkit will allow multiple nucleic acids to be tested at once, giving an output based on a specific set of inputs leading to faster turnaround times on pathogen detection. Fig 1A shows the simplest form of such an assay for which we have shown nucleic acid detection down to pM levels.

Figure 3. Nucleic acid biosensing. In its simplest form can be formed from 1-2 labeled capture probes encoding the reverse complement of the target sequence.
  1. Lymperopoulos K*, Crawford R*, Torella JP, Heilemann M, Hwang LC, Holden SJ, and Kapanidis AN, Single-molecule DNA biosensors for protein and ligand detection. Angew. Chem. 2010; 49 (7), pp.1316-1320. *equal contribution.
  2. Crawford R, Kelly DJ, Kapanidis AN, A Protein Biosensor That Relies on Bending of Single DNA Molecules, ChemPhysChem 2012; 13 (4), pp.918-922.

2. DNA as a Photonic Waveguide

David Bauer, Achillefs Kapanidis

Light-harvesting complexes found in nature are vivid examples of nanoscale photonic waveguides. After an absorption of a photon by pigment-protein complexes, energy is transfered through a series of radiationless transfer to the transmembrane reaction centre complex. DNA is a good candidate material for such photonic wires, mainly due to the availability of several labelling strategies for introducing chromophores on DNA (Refs. 1-3). We are currently working on improving the generality and transfer efficiency of DNA photonic wires by using (1) homogeneous repeat elements; (2) a cascade of spectrally different fluorophores; and (3) non-specific fluorophores as energy transfer intermediates.


A nanometer-sized molecular photonic wire based on DNA as rigid scaffold (upper panel), along with the absorption and emission spectra of the fluorophore used (lower panel). (Tinnefeld et al., 2005)
  1. Heilemann M, Tinnefeld P, Mosteiro GS, Parajo MG, Van Hults NG, and Sauer M, Multistep energy transfer in single molecular photonic wires. J. Am. Chem. Soc. 2004; 126, pp.6514-6515.
  2. P. Tinnefeld, M. Heilemann, and M. Sauer. Design of molecular photonic wires based on multistep electronic excitation transfer. Chem. Phys. Chem. 2005; 6, pp.217-222.
  3. S. Vyawahare, S. Eyal, K.D. Mathews, and S.R. Quake. Nanometer-scale fluorescence resonance optical waveguides. Nano lett. 2004; 4, pp.1035-1039.

3. DNA cages for transcription factors

Robert Crawford, Achillefs Kapanidis

This project is a collaboration with The DNA group led by Prof Andrew Turberfield (also Oxford Physics Link) and extends successful work on a DNA tetrahedron [1]. In this case we harness the self-assembly properties of DNA to produce a cage that encapsulates the transcription factor catabolite activator protein (Fig 1; [1]). Such a protein encapsulating cage could be used as a drug delivery vehicle or for protein structural studies using FRET. We prove encapsulation and correct orientation of CAP within the cage using single-molecule FRET and demonstrate the effect of the cage at slowing the off-rate of CAP compared to a linear sequence.


Figure 1. A nanoscale cage designed to encapsulate the transcription factor catabolite activator protein.
  1. Goodman RP, Heilemann M, Doose S, Erben CM, Kapanidis AN, Turberfield AJ, Reconfigurable, braced, three-dimensional DNA nanostructures. Nat Nanotechnol. 2008; 3(2), pp.93-6.
  2. Crawford R, Erben CM, Periz J, Hall LM, Brown T, Turberfield AJ, and Kapanidis AN, Non-covalent Single Transcription Factor Encapsulation Inside a DNA Cage. Angew. Chem. (accepted, forthcoming).