Professor, Imperial College, 2012
Associate Professor, Wayne State Univerisity, 2011-12
Assistant Professor, Wayne State Univerisity, 2005-11
Postdoctoral Fellow, Univeristy of Michigan, 2001-05
Docteur ès Sciences, EPF Lausanne, 2001
Dipl. Chem. Eng., EPF Lausanne, 1997
Research in the Rueda lab involves the development of quantitative single-molecule approaches to investigate the mechanism of complex biochemical systems (incl. RNA, DNA and protein). Single molecule microscopy has opened up new avenues leading to important discoveries on how structural dynamics correlate to the function of nucleic acids and proteins. An attractive aspect of single-molecule microscopy is that it reveals the structural dynamics of individual molecules, otherwise hidden in ensemble-averaged experiments, thereby providing direct observation of key reaction intermediates (even low populated or short lived ones) and the characterization of reaction mechanisms.
- RNA folding: RNA molecules play numerous essential roles in living cells. In addition to their well-known function as information carriers, they can also participate in catalysis (e.g., splicing, translation, etc) and control of gene expression (e.g., siRNA, riboswitches, etc). The discovery of different functional aspects of RNA molecules has increased their potential applications in medicine and biotechnology. The folding of RNA into precise three-dimensional structures is essential for proper function in vivo. Although RNA molecules make use of only four nucleobases, their ability to fold into a seemingly infinite number of dynamic structures is key for their functional diversity. Using single molecule microscopy among other biophysical tools, we explore the fundamental principles that govern RNA folding from individual folding motifs to large, multidomain, catalytic RNAs. In addition, we also study how remodeling proteins, such as RNA helicases, assist in this process under physiological conditions.
- RNA Splicing: Splicing is an essential step in the maturation of eukaryotic pre-mRNAs. Anomalous pre-mRNA splicing can have lethal effects for the cell and has been linked to numerous diseases such as breast, colorectal, epithelial and ovarian cancers, as well as neurodegenerative disorders such as Parkinson’s and Alzheimer’s. The spliceosome is a large dynamic assembly of 5 small nuclear RNAs (snRNA) and over 100 proteins that catalyzes splicing. It undergoes several, highly conserved, conformational rearrangements. The active site of the spliceosome comprises two key snRNAs (U2 and U6) that have been shown to undergo splicing-related catalysis in absence of proteins. The structure and dynamics of the U2-U6 complex are thought to play critical roles in the mechanism of splicing in vivo. Using active spliceosomes in yeast cell extracts reconstituted with flurophore-labeled U6 snRNA, we explore the role of these dynamics in splicing assembly and catalysis.
- DNA Replication: The catalytic mechanism of DNA polymerases involves multiple steps that precede and follow the transfer of a nucleotide to the 3’-hydroxyl of the growing DNA chain. However, the mechanism by which they achieve their extraordinary accuracy remains unclear. Specifically, kinetic intermediates involved in proofreading have never before been characterized. We use single-molecule approaches to monitor the movement of E. coli DNA polymerase I (Klenow fragment) on a DNA template during DNA synthesis with single base-pair resolution.
- ssDNA Scanning and Deamination: The APOBEC family of enzymes comprises single-stranded ssDNA cytosine deaminases that play important roles in eliminating retroviral infectivity and somatic hypermutation (SHM). For example, APOBEC3G eliminates HIV1 infectivity by converting C→U in numerous small target motifs on the minus viral cDNA, wheras AID generates advantageous mutations in the variable region of immunoglobulin genes in B-cells that increase the affinity of antibodies for antigenes. Compared with dsDNA scanning enzymes (e.g., DNA glycosylases) that excise rare aberrant bases, there is a paucity of mechanistic studies on ssDNA scanning enzymes. We investigate ssDNA scanning and motif-targeting mechanisms for Apo3G and AID using single molecule fluorescence. We address specific issues of deamination activity within the general context of ssDNA scanning mechanisms.
et al., 2019, DNA stretching induces Cas9 off-target activity, Nature Structural and Molecular Biology, Vol:26, ISSN:1545-9985, Pages:185-192
et al., DNA stretching induces Cas9 off-target binding and cleavage, Nature Structural and Molecular Biology, ISSN:1545-9985
et al., 2018, Optimal molecular crowding accelerates group II intron folding and maximizes catalysis, Proceedings of the National Academy of Sciences, Vol:115, ISSN:0027-8424, Pages:11917-11922
et al., 2018, Reduced structural flexibility for an exonuclease deficient DNA polymerase III mutant, Physical Chemistry Chemical Physics, Vol:20, ISSN:1463-9076, Pages:26892-26902
et al., 2018, Structure and dynamics of the yeast SWR1:nucleosome complex, Science, Vol:362, ISSN:0036-8075