Biology subjects

Joo, C.

Publications and source records attributed to Joo, C..

4 recordsLinked to original sources

bioRxiv2018

CRISPR Cas9 searches for a protospacer adjacent motif by one-dimensional diffusion

Since its discovery, the CRISPR/Cas9 system has been at the focus of fundamental researchers, genome engineers, and the general public alike. Despite being in the spotlight for several years, aspects of the precise molecular mechanism of Cas9 activity remain ambiguous. We use single-molecule Foerster resonance energy transfer (smFRET) to reveal Cas9 target search mechanism with nanometer sensitivity. We have developed single-molecule assays to monitor transient interactions of Cas9 and DNA in real time. Our study shows that Cas9 interacts with the protospacer adjacent motif (PAM) sequence weakly, yet probing neighboring sequences via lateral diffusion. This dynamic mode of interactions leads to translocation of Cas9 to another PAM nearby and consequently an on-target sequence. We propose a model in which lateral diffusion competes with 3-dimensional diffusion and thus might aid PAM finding and consequently on-target binding.

biophysics↗

bioRxiv2018

Multiplex single-molecule DNA barcoding using an oligonucleotide ligation assay

Detection of specific nucleic acid sequences is invaluable in biological studies such as genetic disease diagnostics and genome profiling. Here we developed a highly sensitive and specific detection method that combines an advanced oligonucleotide ligation assay (OLA) with multicolor single-molecule fluorescence. We demonstrated that 7-nt long DNA barcodes have the optimal short length to ascertain specificity while being long enough for sufficient ligation. Using four spectrally separated fluorophores to label DNA barcodes, we simultaneously distinguished four DNA target sequences differing by only a single nucleotide. Our new single-molecule approach will allow for accurate identification of low abundance molecules without the need for target DNA pre-amplification.

biophysics↗

bioRxiv2017

Single-Molecule Peptide Fingerprinting

Proteomic analyses provide essential information on molecular pathways of cellular systems and the state of a living organism. Mass spectrometry is currently the first choice for proteomic analysis. However, the requirement for a large amount of sample renders a small-scale proteomics study, such as single-cell analysis, challenging. Here we demonstrate a proof of concept of singlemolecule FRET-based protein fingerprinting. We harnessed the AAA+ protease ClpXP to scan peptides. By using donor fluorophore-labeled ClpP, we sequentially read out FRET signals from acceptor-labeled amino acids of peptides. The repurposed ClpXP exhibits uni-directional processing with high processivity and has the potential to detect low-abundance proteins. Our technique is a promising approach for sequencing protein substrates using a small amount of sample.\n\nSIGNIFICANCEProtein sequencing remains a challenge for small samples. A sensitive sequencing technology will create the opportunity for single-cell proteomics and real-time screening for on-site medical diagnostics. In order to resolve protein identity, we previously developed a computational algorithm that analyzes the ordered sequence of only two types of amino acids within a protein species. Through modification of a biological nanomachine, here we developed single-molecule fluorescence technology to linearize protein molecules and to read signals from labeled amino acids in an ordered manner. This proof of concept of singlemolecule fingerprinting will open a new door to single-molecule protein sequencing and pave the road towards the development of a new, fast, and reliable diagnostic tool.

biophysics↗

bioRxiv2017

Repetitive DNA reeling by the Cascade-Cas3 complex in nucleotide unwinding steps

CRISPR-Cas loci provide an RNA-guided adaptive immune system against invading genetic elements. Interference in type I systems relies on the RNA-guided surveillance complex Cascade for target DNA recognition and the trans-acting Cas3 helicase/nuclease protein for target degradation. Even though the biochemistry of CRISPR interference has been largely covered, the biophysics of DNA unwinding and coupling of the helicase and nuclease domains of Cas3 remains elusive. Here we employed single-molecule FRET to probe the helicase activity with a high spatiotemporal resolution. We show that Cas3 remains tightly associated with the target-bound Cascade complex while reeling in the target DNA using a spring-loaded mechanism. This spring-loaded reeling occurs in distinct bursts of three base pairs, that each underlie three successive 1-nt unwinding events. Reeling is highly repetitive, compensating for inefficient nicking activity of the nuclease domain. Our study reveals that the discontinuous helicase properties of Cas3 and its tight interaction with Cascade ensure well controlled degradation of target DNA only.

biophysics↗