Biology subjects

Luk, K.

Publications and source records attributed to Luk, K..

20 records · Page 2Linked to original sources

bioRxiv2020

Potent inhibitors of toxic alpha-synuclein oligomers identified via cellular time-resolved FRET biosensor.

Preventing or reversing the pathological misfolding and self-association of alpha-synuclein (aSyn) can rescue a broad spectrum of pathological cellular insults that manifest in Parkinsons Disease (PD), Dementia with Lewy bodies (DLB), and other alpha-synucleinopathies. We have developed a high-throughput, FRET-based drug discovery platform that combines high-resolution protein structural detection in living cells with an array of functional and biophysical assays to identify novel lead compounds that protect SH-SY5Y cells from aSyn induced cytotoxicity as well as inhibiting seeded aSyn aggregation, even at nanomolar concentrations. Our combination of cellular and cell-free assays allow us to distinguish between direct aSyn binding or indirect mechanisms of action (MOA). We focus on targeting oligomers with the requisite sensitivity to detect subtle protein structural changes that may lead to effective therapeutic discoveries for PD, DLB, and other alpha-synucleinopathies. Pilot high-throughput screens (HTS) using our aSyn cellular FRET biosensors has led to the discovery of the first nanomolar-affinity small molecules that disrupt toxic aSyn oligomers in cells and inhibit cell death. Primary neuron assays of aSyn pathology (e.g. phosphorylation of mouse aSyn PFF) show rescue of pathology for two of our tested compounds. Subsequent seeded thioflavin-t (ThioT) aSyn aggregation assays demonstrate these compounds deter or block aSyn fibril assembly. Other hit compounds identified in our HTS are known to modulate oxidative stress, autophagy, and ER stress, providing validation that our biosensor is sensitive to indirect MOA as well.

neuroscience↗

bioRxiv2019

Efficient Homology-directed Repair with Circular ssDNA Donors

While genome editing has been revolutionized by the advent of CRISPR-based nucleases, difficulties in achieving efficient, nuclease-mediated, homology-directed repair (HDR) still limit many applications. Commonly used DNA donors such as plasmids suffer from low HDR efficiencies in many cell types, as well as integration at unintended sites. In contrast, single-stranded DNA (ssDNA) donors can produce efficient HDR with minimal off-target integration. Here, we describe the use of ssDNA phage to efficiently and inexpensively produce long circular ssDNA (cssDNA) donors. These cssDNA donors serve as efficient HDR templates when used with Cas9 or Cas12a, with integration frequencies superior to linear ssDNA (lssDNA) donors. To evaluate the relative efficiencies of imprecise and precise repair for a suite of different Cas9 or Cas12a nucleases, we have developed a modified Traffic Light Reporter (TLR) system [TLR-Multi-Cas Variant 1 (MCV1)] that permits side-by-side comparisons of different nuclease systems. We used this system to assess editing and HDR efficiencies of different nuclease platforms with distinct DNA donor types. We then extended the analysis of DNA donor types to evaluate efficiencies of fluorescent tag knock-ins at endogenous sites in HEK293T and K562 cells. Our results show that cssDNA templates produce efficient and robust insertion of reporter tags. Targeting efficiency is high, allowing production of biallelic integrants using cssDNA donors. cssDNA donors also outcompete lssDNA donors in template-driven repair at the target site. These data demonstrate that circular donors provide an efficient, cost-effective method to achieve knock-ins in mammalian cell lines.

molecular biology↗