Revolutionizing Single-Molecule Fluorescence Experiments

Biomolecular behavior is intricately complex, with structural and functional information often eluding traditional biochemistry methods. The functionality of biopolymers such as DNA, RNA, and proteins heavily depends on the sequence of their respective building blocks. However, conventional single-molecule methods are constrained by time and cost, limiting the number of sequences that can be studied as each sequence requires separate preparation and measurement.

In response to this challenge, we have developed an innovative method called Single-molecule Parallel Analysis for Rapid eXploration of Sequence space (SPARXS). This novel approach integrates ensemble fluorescence experiments with next-generation sequencing, performing millions of parallel ‘single-molecule’ fluorescence experiments for thousands of sequences simultaneously. This high-throughput method allows for the acquisition of diverse sequence-dependent biophysical and biochemical properties at the single-molecule level.

The broad applicability of SPARXS and the wealth of information it provides will significantly advance our understanding of how sequence influences molecular structure and function. This new dimension in single-molecule experiments will have profound implications for understanding biological mechanisms, enabling the development of more accurate models that may ultimately lead to groundbreaking advancements in biomolecular engineering. This project was supported by ERC Consolidator (2018-2024).

Developing Single-Molecule Protein Sequencing Techniques

Proteins are vital in all biological systems as they are the working machineries of cells. There are >20,000 protein species inside human cells which are expressed at all different levels. Medical scientists read the amino acid sequences of proteins to analyze the protein expression profiles of human cells; and biologists to chart protein‐protein interaction maps. Complete mapping, however, has not been achieved since current sequencing techniques have intrinsic limitations.

        We aim to develop several single-molecule sequencing techniques in response to urgent demand for a new large-scale, highly sensitive, error-free method (see figure). This new approach will probe biological objects, molecule by molecule, not just take their average. This approach will cover an entire population despite the complex nature and the wide dynamic range of cellular objects. Unlike conventional sequencing, this sequencing will be less error-prone for its direct measurement. Single-molecule detection is so sensitive that this approach will require only a small amount of sample (no more than 1 fmol) for the analysis of cellular objects. This will create the opportunity for single-cell analysis. This novel sequencing approach will change the paradigm of sequencing techniques. This project is supported by NWO Vrije FOM Programme (2017-2021) and NWO talent program, Vici (2021-2026), and is conducted in collaboration with five research groups who bring expertise of chemical engineering, bioinformatics, nanopores, fluorescence, and molecular biology. 

Artist's impression of single-molecule protein sequencing

Harnessing the Genome Editing Ability of Bacteria for Genome Editing

Genome editing is an essential tool for life sciences. Breakthroughs in 2012-2013 drew our attention to the genome editing ability of bacteria. We aim to understand this remarkable feature and to harness it for applications in science, technology, and society.

    Bacteria use a remarkable genome editing strategy to win over invading genetic elements. When viruses invade, bacteria allocate a part of their genome (CRISPR) to store the genetic material from viruses. When a known virus returns, bacteria recognize the invading DNA using this memory and eliminate it. Recent research has led to fascinating genetic and biochemical insights on this CRISPR immune system. However, the molecular mechanism of CRISPR remains poorly understood. We use multi-color single-molecule fluorescence techniques to study the molecular mechanism of the CRISPR system. We investigate how a bacterium acquire genetic material from viruses, interrogate and destroy viral DNA targets, and reprogram their memory. Utilizing high spatio-temporal resolution of single-molecule approaches, we aim to observe these processes in real time and quantitatively examine their dynamics. We apply the outcome of this study to investigate the genome editing tool, S. pyogenes Cas9. This project was supported by NWO Vidi (2015-2019). 

CRISPR for genome engineering (figure from a cover of Nature, 2017)