Improved synthesis and application of an alkyne-functionalized isoprenoid analogue to study the prenylomes of motor neurons, astrocytes and their stem cell progenitors.

Protein prenylation is one example of a broad class of post-translational modifications where proteins are covalently linked to various hydrophobic moieties. To globally identify and monitor levels of all prenylated proteins in a cell simultaneously, our laboratory and others have developed chemical proteomic approaches that rely on the metabolic incorporation of isoprenoid analogues bearing bio-orthogonal functionality followed by enrichment and subsequent quantitative proteomic analysis. Here, several improvements in the synthesis of the alkyne-containing isoprenoid analogue C15AlkOPP are reported to improve synthetic efficiency. Next, metabolic labeling with C15AlkOPP was optimized to obtain useful levels of metabolic incorporation of the probe in several types of primary cells. Those conditions were then used to study the prenylomes of motor neurons (ES-MNs), astrocytes (ES-As), and their embryonic stem cell progenitors (ESCs), which allowed for the identification of 54 prenylated proteins from ESCs, 50 from ES-MNs, and 84 from ES-As, representing all types of prenylation. Bioinformatic analysis revealed specific enriched pathways, including nervous system development, chemokine signaling, Rho GTPase signaling, and adhesion. Hierarchical clustering showed that most enriched pathways in all three cell types are related to GTPase activity and vesicular transport. In contrast, STRING analysis showed significant interactions in two populations that appear to be cell type dependent. The data provided herein demonstrates that robust incorporation of C15AlkOPP can be obtained in ES-MNs and related primary cells purified via magnetic-activated cell sorting allowing the identification and quantification of numerous prenylated proteins. These results suggest that metabolic labeling with C15AlkOPP should be an effective approach for investigating the role of prenylated proteins in primary cells in both normal cells and disease pathologies, including ALS.

Combined recombinase polymerase amplification/rkDNA-graphene oxide probing system for detection of SARS-CoV-2.

The development of rapid, highly sensitive, and selective methods for the diagnosis of infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) should help to prevent the spread of this pandemic virus. In this study, we combined recombinase polymerase amplification (RPA), as a means of isothermal DNA amplification, with an rkDNA-graphene oxide (GO) probe system to allow the rapid detection of SARS-CoV-2 with high sensitivity and selectivity. We used in situ enzymatic synthesis to prepare an rkDNA probe that was complementary to an RPA-amplified sequence of the target N-gene of SARS-CoV-2. The fluorescence of this rkDNA was perfectly quenched in the presence of GO. When the quenched rkDNA-GO system was added to the RPA-amplified sequence of the target SARS-CoV-2, the fluorescence recovered dramatically. The combined RPA/rkDNA-GO system exhibited extremely high selectivity (discrimination factor: 17.2) and sensitivity (LOD = 6.0 aM) for the detection of SARS-CoV-2. The total processing time was only 1.6 h. This combined RPA/rkDNA-GO system appears to be a very efficient and simple method for the point-of-care detection of SARS-CoV-2.