Research

The overall goal of Dr. CHEN’s research group in the School of Life and Health Sciences, School of Medicine, at The Chinese University of Hong Kong, Shenzhen (CUHK-Shenzhen) is to better understand the structures and the physical-chemical properties of RNAs and RNA-ligand complexes to provide deeper insight into and to facilitate precise control of the diverse biological functions involving RNA. We aim to use the fundamental knowledge to fight neurodegenerative diseases, cancers, bacterial and viral infections by designing and discovering novel therapeutic ligands targeting RNA. To approach the challenging goals, we have assembled a multidisciplinary team with expertise ranging from molecular biophysics, structural biology, computation, chemical synthesis, cell biology, animal model studies, to medical healthcare. The research projects of current interests are: (1) characterizing the molecular recognition interactions (e.g., hydrogen bonding and aromatic base stacking) accounting for structure, stability, and dynamics of RNA structural building blocks such as internal loops, hairpins, triplexes, and pseudoknots, (2) probing the complex energy landscapes of RNA folding and assembly with protein, (3) designing and discovering therapeutic ligands (small molecules, oligonucleotides, peptides, peptide nucleic acids, etc.) targeting RNA, (4) developing nucleic acid based nano-biosensors to rapidly detect toxic/pathogenic agents in food products and human body, and (5) discovering and characterizing novel nucleic acid based nano-catalysts for important organic and inorganic reactions at mild conditions.

We employ various conventional and cutting-edge techniques including laser optical tweezers, NMR, UV-Vis absorption, fluorescence, SPR, gel electrophoresis, PCR, chemical synthesis of modified oligonucleotides and peptides, in vitro transcription, protein expression, and cell culture assay. The research experience in the laboratory will help the students to grasp fundamental knowledge and experimental skills, to develop learning skills such as rigorous reasoning and innovative thinking, and to be able to ask and answer important questions within and beyond chemical and molecular sciences.

Research Grants:

Research Grants at CUHK-Shenzhen:

PI, 2022-2025, The National Natural Science Foundation of China, General Program

University Development Fund

External Grants at NTU

PI, Nov 2019 – Oct 2022 (terminated in Sep 2020), Detecting and targeting influenza viral panhandle RNA structure, S$604,730 (with an additional Indirect Cost of S$120,946), MOE AcRF Tier 2

Co-PI, Jan 2019 – Jan 2022, Role of RNA editing in guarding against hepatic injury and hepatocarcinogenesis, S$15,000 (S$687,500), MOE AcRF Tier 2

PI, Jan 2016 – Dec 2018, Targeting pre-mRNA and pre-miRNA by RNA duplex-binding peptide nucleic acids, S$554,327 (with an additional Indirect Cost of S$110,865), MOE AcRF Tier 2

PI, Apr 2014 – Mar 2017, Unravelling mRNA structure’s role in translational regulation, S$542,534 (with an additional Indirect Cost of S$108,507), MOE AcRF Tier 2

Internal Grants at NTU

PI, Apr 2018 – Mar 2019, Developing near-infrared fluorescent light-up probes for detecting RNA structures in viruses and cancers, S$40,000, NTU-A*STAR Seed Funding Research Award

PI, Mar 2018 – Feb 2020, Tagging CRISPR guide RNAs, S$99,999, MOE AcRF Tier 1

PI, Nov 2015 – Nov 2017, Creating high-information-content nano-structures based on modified nucleic acids, S$99,999, MOE AcRF Tier 1

PI, Nov 2013 – Oct 2015, Synthesis of modified triplex-forming peptide nucleic acids for targeting HIV-1 ribosomal frameshift stimulatory RNA structure, S$99,999, MOE AcRF Tier 1

PI, Mar 2011 – Feb 2014, Targeting RNA duplexes with modified triplex-forming oligonucleotides: From atomic mutation to single-molecule manipulation, S$200,000, MOE AcRF Tier 1

Co-PI, Jan 2011 – Dec 2013, Solid state nanopore devices for single molecule biophysics and sequencing, S$25,000 (S$75,000), SPMS Collaborative Research Award

PI, Jul 2010 – Dec 2022, RNA structures, dynamics, and function, S$269,700, Nanyang Technological University Start-Up Grant

Short summary of our research:

RNAs become increasingly important disease biomarkers and drug targets. RNAs often form complex secondary structures containing both single-stranded (ss) loop and double-stranded (ds) stem regions, with the dsRNAs mainly stabilized by Watson–Crick base pairing and stacking interactions. By nano-manipulation using optical tweezers in combination with other biophysical methods, we have revealed at the single-molecule level the contributions of molecular recognition interactions such as a single hydrogen bond in a base pair or base triple to RNA folding and unfolding and characterized the effects of a single proton binding. We have unveiled the correlations (i) between mRNA mechanical properties and mRNA structure induced translational reading frame shifting and (ii) between mechanical properties of tau pre-mRNA splice site hairpin structures and pre-mRNA alternative splicing activities.

In addition, we are developing a molecular recognition platform based on chemically modified Peptide Nucleic Acids (PNAs) for the targeting of RNA structures in a sequence- and structure-specific manner. Specifically, we program our dsRNA-binding PNAs (dbPNAs) with a new four-letter chemical code (T, L or R, E or S, and Q) for the recognition of RNA Watson–Crick duplexes, through the formation of major-groove triples. We have demonstrated that short (e.g., 10-mer) PNAs containing L, R, E, S, or Q show enhanced sequence-specific recognition of RNA base pairs in dsRNAs with significantly weakened binding to ssRNAs or dsDNAs at near-physiological conditions. Our cell culture studies show that our PNAs conjugated with small molecules are bioactive in the targeting of viral and cellular RNAs. Thus, we will further develop our dbPNAs platform as useful diagnostic tools and therapeutic drugs to fight against viral infections, cancers, and neurodegenerative diseases.

Our research contribution and impact:

Chemically-modified short (e.g., 10-mer) PNA oligomers that sequence-specifically bind to dsRNAs are promising complementary tools of traditional antisense strands and small molecules for viral inhibition and regulation of cellular functions. The dsRNA regions are often relatively rigid and may not be easily accessible by traditional antisense oligonucleotides and small molecules, which are usually ideal for targeting non-Watson-Crick regions such as junctions, bulges, and internal loops. For example, formation of PNA·dsRNA triplexes has been shown to result in the selective inhibition of translation (work by Eriks ROZNERS lab from Binghamton University, USA) and ribosomal frameshifting (work by our lab), respectively, through steric blockage and/or stabilization of RNA duplex structures. The relatively short length (10-mer) of our dsRNA-binding PNA may also minimize the solubility problem observed for some longer PNAs.

The dsRNA-binding PNAs platform is programmable and may allow us to design PNAs binding to many functionally important viral and cellular RNA structures. Our research will have a significant impact on the field of the RNA interactome, specifically pertaining to the identification and targeting of dsRNA structures important in RNA biology and pathology. Further demonstration of the applications our PNAs platform in the targeting of various biomedically important RNA structures in cell cultures and animal models will help establish industrial collaborations to translate our laboratory ideas into real products.

Our high-impact research on targeting RNA duplexes has generated significant interests in the RNA community. We wrote an invited review article on RNA triplexes in the journal Wiley Interdisciplinary Reviews: RNA. We also wrote an invited book chapter (published by Springer) on recognition and targeting of mature miRNA and miRNA hairpin precursor by duplex and triplex formation, respectively. We were also invited to write a methods article for the Journal of Visualized Experiments on sequence-specific and selective recognition of double-stranded RNAs over single-stranded RNAs by chemically modified peptide nucleic acids. We wrote a News & Views article for the journal Nature Chemical Biology on the importance of base triples, junctions, and other non-Watson-Crick interactions in facilitating catalytic reactions of an RNA enzyme. We were invited by the editor-in-chief of Biochemistry (Alanna Schepartz from Yale University, USA) to contribute a paper to the Future of Biochemistry special issue in Jan 2018. I have been also invited to give keynote and invited talks at conferences.

We have secured three MOE Tier 2 grants for the biophysical characterization and application studies of our chemically-modified PNAs. Furthermore, I’m a co-principal investigator for a MOE Tier 2 grant (01/2019-01/2022, PI: Polly Leilei CHEN, National University of Singapore, Project title: Role of RNA editing in guarding against hepatic injury and hepatocarcinogenesis).

The research has been mainly carried out by postdoctoral research fellows and graduate students. Many undergraduate and pre-university students have also been involved in the research. The poster and oral presentations of the staff and students have also been recognized with awards at various conferences and scientific festivals (http://www.ntu.edu.sg/home/rnachen/members.htm ).