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)

Repurposing the DNA Elimination for Genome Editing

CRISPR has revolutionized the way of editing a genome. Despite its wide use, CRISPR-genome editing has limitations, especially in the use for medical applications. Numerous studies have shown that 

it suffers from the off-target effect. Its use is also restricted by its particular sequence requirement and its poor accessibility to a structured genome. Furthermore, recent studies suggested that it might act as a virulence factor within human cells. These limitations demand new genome editing tools.

        We aim to understand the molecular mechanism of Tetrahymena DNA elimination. This naturally occurring genome editing is mediated by a eukaryotic RNA system (Twi1).  This system uses an entirely different mechanism from CRISPR and has potential to perform more effectively. We investigate how small RNA-loaded Twi1 (“target searcher”) recognizes its target and whether its performance exceeds other target searchers including CRISPR/Cas9. We use single-molecule fluorescence for high resolution observations and develop a high-throughput single-molecule method for transcriptome-wide understanding. We further aim to identify a Twi1-related DNA nuclease(s) that carries out DNA elimination. We use cutting-edge tools of single-molecule pull-down and multi-color FRET together with mass spectrometry. The nanoscopic understanding of a searcher (Twi1) and the identification of a nuclease will help create a new genome editing tool (e.g. a fusion of Twi1 and the nuclease) that potentially perform better than Cas9. Thereby, this fundamental study will make a long-term impact for applications in science and technology. This project is supported by ERC (2019-2023). (Figure from Hamilton et al, eLife, 2016)

DNA elimination in a ciliated protozoan