Research

Single-Molecule Protein Sequencing
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 a single-molecule sequencing technique in response to urgent demand for a new large-scale, highly sensitive, error-free method. 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. Our patent was published. For more information, contact us. (Figure modified from "Alphabet New and Old")

Anti-Viral Defense to 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 single-molecule fluorescence, multi-color FRET (Forster Resonance Energy Transfer) techniques to study the molecular mechanism of the CRISPR system. We investigate how a model organism, E. coli, 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 will apply the outcome of this study to investigate the genome-editing tool, S. pyogenes Cas9. (Figure by Andreus | Dreamstime.com)
        Gedreven evolutionaire wapenwedloop zijn bacteriën in staat om hun genoom te modificeren. Door gebruik te maken van geavanceerde microscopie zal Dr. C. (Chirlmin) Joo aan de TU Delft deze genetisch veranderende eigenschappen zichtbaar maken op een moleculair niveau. De kennis die hij uit deze studie vergaard zal gebruikt kunnen worden voor het ontwikkelen van een betrouwbare genetische modificatie techniek met vele toepassingen in de biotechnologie.

Gene Silencing to Gene Regulation
MicroRNA (miRNA) is a short, single-stranded RNA of ~22 nucleotides. This non-coding RNA works as a regulatory component in eukaryotes, controlling embryonic development, apoptosis, tumorigenesis, anti-viral defense, and other cellular functions. This regulation process (termed RNA interference or RNAi) occurs when a microRNA-loaded RISC (RNA-induced silencing complex) binds to a target mRNA. A microRNA acts to guide the RISC by base-pairing with a target mRNA.
        When small RNAs are exogenously introduced into cells, microRNA machineries are hijacked, and the exogenous small RNAs trigger artificial RNAi. The mechanism of action of RNAi is sequence-specific, and thus endogenous genes can be manipulated using custom-designed small RNAs (termed small interference RNAs or siRNAs). This finding has introduced the possibility of using siRNAs for biomedical applications. With the human genome available in hand and RNA synthesis easily accessible, small RNA-based therapeutics have been instigated.
        We investigate the whole spectrum of small RNA biogenesis, action, and regulation. We utilize the cutting edge technologies including single-molecule fluorescence, multi-color FRET, and single molecule immunoprecipitation. The knowledge we obtain from these small RNA studies will be applied to the development of small RNA-based therapeutics. (Figure from sprna.com)
        
Het centrale dogma van moleculaire biologie stelt dat genetische informatie van DNA wordt omgezet naar eiwitten en in dit proces werd RNA gezien als een passieve drager van informatie. Recente baanbrekende ontdekkingen hebben onze visie sterk doen veranderen, waarbij RNA van een passieve drager van informatie is veranderd naar een belangrijk element in cel regulatie. Ons laboratorium doet onderzoek aan microRNAs, dit zijn kleine stukjes RNA die bijna alle mRNAs in eukaryote cellen reguleren. Wij gebruiken een single-molecule fluorescentie techniek om aan te tonen hoe deze microRNAs worden gegenereerd en hoe ze hun doelwit op het mRNA vinden. Wij gebruiken een liniaal op nanometer schaal, FRET (fluorescence resonance energy transfer), om deze moleculaire processen te ontleden en te kwantificeren. Wij hebben een multicolor single molecule fluorescentie techniek toegepast om meerdere componenten tegelijk zichtbaar te maken. Daarnaast hebben we recent een techniek ontwikkeld waarmee we microRNA eiwit complexen kunnen reconstrueren met behulp van immunoprecipitatie. Deze eerste single-molecule studie op microRNAs zal de moleculaire mechanismen van microRNA gereguleerde gen expressie onthullen. De vruchtbare uitkomst van dit onderzoek zal in de nabije toekomst een rol gaan spelen in de ontwikkeling van nieuwe RNA gebaseerde gen therapieën.


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