Research

The workings of living systems are the result of the rich physics that emerges from their nanoscopic building blocks. This intriguing nanoscopic world is becoming increasingly accessible to quantitative observation and nanomanipulation techniques. We aim to explore and exploit the physics of biomolecular systems such as DNA and molecular motors using quantitative experimental analysis and modeling at the single-molecule level. In our research endeavors we develop innovative biophysical research methodologies and push the limits of quantitative experimental analysis and (nanoscale) imaging methods.

My research is motivated by the drive to:

elucidate and quantify the molecular physics of essential biological processes
push the limits of our ability to explore the biomolecular world
exploit biophysical principles to create new research methods and devices
link research fields through innovative methodology

Current reseach topics

Physics of ice binding proteins and crystal growth
Biophysics of DNA and DNA-processing proteins
Analysis of DNA replication, transcription, repair, and compaction
Developing new and/or enhanced quantitative biophysical methods based on force spectroscopy, microfluidics and (super-resolution) microscopy

Current reseach topics

Physics of ice binding proteins and crystal growth

Growth of ice crystals can induce critical damage to soft condensed matter systems such as living tissue and cells. Many lifeforms produce ice-binding proteins (IBPs) for protection against frost-damage at temperatures as low as -70C. It is yet unclear how IBPs can function as such remarkable cryoprotectants. New insights into the biophysics of ice-binding proteins can help to harness and optimize their cryoprotective potential in e.g. healthcare, crop protection, food technology, and anti-icing approaches.

IBPs form a diverse group of proteins with common features of binding ice at specific crystal planes and protecting against freeze damage. Two major cryoprotective functions can be identified: freeze-avoiding function by preventing growth of ice crystals to several degrees below the melting point, or freeze-tolerating function by keeping ice grains small below freezing. These functions are termed the thermal hysteresis effect (TH, figure right, first panel) and ice recrystallization inhibition effect (IRI, figure right, second panel), respectively.

Although models exist that relate IBP activity to pinning of advancing ice planes (see image right), numerous open questions regarding the microscopic mechanisms remain. We search for answers to these mechanistic questions by probing IBP activity at the molecular length scales where IBPs act. To this end, we are developing innovative single-molecule methodology based on optical tweezers, (super-resolution) fluorescence microscopy and novel refrigeration methods to directly resolve dynamic interactions between ice, water, and proteins on the nanometer length scale.

Biophysical methods developments

Our knowledge of the building blocks of life has advanced significantly through the ongoing development of single-molecule biophysical methods. We push the limits of our ability to explore the biomolecular world by developing new and advanced single-molecule approaches. Such development is done with specific biological questions or experimental challenges in mind. The combination of optical trapping and fluorescence microscopy is for example particularly powerful for quantitative analyses of biomolecular systems such as DNA-protein complexes.

Single-molecule analysis of DNA compaction, repair, replication and transcription

In several collaborations we have exploited the use of optical tweezers and fluorescence microscopy to analyze the molecular mechanisms of DNA transactions. This includes analysis of DNA replication, DNA compaction, DNA repair and DNA transcription.

Brouwer, I. et al. Sliding sleeves of XRCC4–XLF bridge DNA and connect fragments of broken DNA. Nature 535, 566–569 (2016).

Farge, G. et al. Protein sliding and DNA denaturation are essential for DNA organization by human mitochondrial transcription factor A. Nat. Commun. 3, 1013 (2012).

Candelli, A. et al. Visualization and quantification of nascent RAD51 filament formation at single-monomer resolution. Proc. Natl. Acad. Sci. 111, 15090–15095 (2014).

Syrjänen, J. L. et al. Single-Molecule observation of DNA compaction by meiotic protein SYCP3. Elife 6, 1–14 (2017).

Optical trapping and fluorescence microscopy

Optical tweezers exploit the momentum of photons to trap microscopic beads and manipulate tethered DNA molecules. Their subpiconewton force-resolution and subnanometer distance-resolution provides access to the most important biological energy- scale: kBT~pN∙nm. This provides extensive opportunities to mechanically control and interrogate DNA-protein complexes. The global mechanical information provided by optical tweezers is exquisitely complemented by the local and specific information provided by fluorescence microscopy: wide-field fluorescence microscopy or beam-scanning confocal microscopy can reveal binding and local activity of labelled proteins along stretched DNA molecules in real-time.

Heller, I., Hoekstra, T. P., King, G. a, Peterman, E. J. G. & Wuite, G. J. L. Optical tweezers analysis of DNA-protein complexes. Chemical Reviews 114, 3087–3119 (2014).

Super-resolution fluorescence microscopy

Dense coverage of DNA by proteins is a ubiquitous feature of cellular processes such as DNA organization, replication, and repair. Super-resolution fluorescence microscopy has prepared the ground for carrying out single-molecule experiments at such higher molecular density. Quantitative experimental access to biologically relevant protein densities is essential for linking idealized in vitro experiments with the realistically crowded in vivo situation in cells.
STED. We previously combined one-dimensional Stimulated Emission Depletion (STED) nanoscopy with optical tweezers. This allows one to hold single DNA strands and image proteins on the DNA at a resolution better than 50 nm and visualize local spatial dynamics at high temporal resolution (better than 50 ms). Current efforts include implementation of 3D STED capable of revealing the detailed architecture of spatially extended structures such as chromosomes and cells.
Localization Microscopy Super-resolution microscopy based on localization microscopy allows reconstructing a super-resolved image from a series of diffraction limited images in which only sparse subpopulations of the emitters fluoresce. DNA can for example be super-resolved by localizing individual binding events of DNA intercalators (Binding-activated localization microscopy). We combined such localization microscopy with optical trapping of DNA (image right). We exploited this principle to develop a new method to super-resolve unlabeled proteins on DNA (see below).

Heller, I. et al. STED nanoscopy combined with optical tweezers reveals protein dynamics on densely covered DNA. Nat. Methods 10, 910–6 (2013).

Heller, I. et al. Mobility Analysis of Super-Resolved Proteins on Optically Stretched DNA: Comparing Imaging Techniques and Parameters. Chemphyschem 15, 727–33 (2014).

Meijering, AEC et al. Imaging unlabeled proteins on DNA with super-resolution. Nucleic Acids Research, 48 e34 (2020)

New methods based on DNA intercalation

DNA intercalators are widely used as fluorescent probes to visualize DNA and DNA transactions in vivo and in vitro. We are developing and exploring several new methods based on DNA intercalation.

Previously, we combined single-dye fluorescence microscopy with force spectroscopy to elucidate the tension-dependent DNA-binding kinetics of a range of commercially available mono- and bis-intercalating cyanine dyes (SYTOX Orange, SYTOX Green, SYBR Gold, YO-PRO-1, YOYO-1 and POPO-3). We showed that optimizing these rates minimizes the perturbing impact of intercalators on, for example, enzymatic activity. These new insights provide handles for the improved use of intercalators as DNA probes with minimal perturbation and/or drugs with maximal efficacy.

Biebricher, A. S. et al. The impact of DNA intercalators on DNA and DNA-processing enzymes elucidated through force-dependent binding kinetics. Nat. Commun. 6, 7304 (2015).

Current projects include the development of several new biophysical methods based on DNA intercalation. This includes the demonstration that DNA intercalators can be exploited for super-resolution imaging of unlabeled proteins on DNA and for quantitative imaging of local tension profiles.

Meijering, AEC et al. Imaging unlabeled proteins on DNA with super-resolution. Nucleic Acids Research, 48 e34 (2020)

King, G. A., Biebricher, A. S., Heller, I., Peterman, E. J. G. & Wuite, G. J. L. Quantifying Local Molecular Tension Using Intercalated DNA Fluorescence. Nano Lett. 18, 2274–2281 (2018).

DNA conformation and structural transitions probed by intercalators

In living cells, the structure of DNA is continuously impacted by biochemical and mechanical cues. Because the local structure and conformation of DNA has a large impact on the binding characteristics of intercalators to DNA, DNA intercalators are excellent fluorescent probes to image structural transitions in DNA. Building on this work, we were able to use intercalators to confirm the existence of a new, hyperstretched state of DNA that we predicted through statistical mechanical modeling.

Schakenraad, K. et al. Hyperstretching DNA. Nat. Commun. 8, 2197 (2017).

Backer, AS et al. Single-molecule polarization microscopy of DNA intercalators sheds light on the structure of S-DNA. Science Advances, 5 eaav1083 (2019)

Backer, AS et al. Elucidating the Role of Topological Constraint on the Structure of Overstretched DNA Using Fluorescence Polarization Microscopy. J. Phys. Chem. B, 125 (3) 8351-8361 (2021)

Meijering, AEC et al. Nonlinear mechanics of human mitotic chromosomes. Nature, 605 545 (2022)

Previous reseach topics

Field-effect transistors made of single carbon nanotubes and graphene
electrochemistry at single nanotube electrodes
electrostatic and electrochemical detection of single biomolecules and cells

Research

My research is motivated by the drive to:

Current reseach topics

Welcome to the Heller lab

DNA repair elucidated

Hyperstretching DNA

Dynamics of DNA-bound proteins

Nanobiotechnology