3 research outputs found
Profiling Esterases in <i>Mycobacterium tuberculosis</i> Using Far-Red Fluorogenic Substrates
Enzyme-activated, fluorogenic probes
are powerful tools for studying
bacterial pathogens, including <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>). In prior work, we reported two 7-hydroxy-9<i>H</i>-(1,3-dichloro-9,9-dimethylacridin-2-one) (DDAO)-derived
acetoxymethyl ether probes for esterase and lipase detection. Here,
we report four-carbon (C4) and eight-carbon (C8) acyloxymethyl ether
derivatives, which are longer-chain fluorogenic substrates. These
new probes demonstrate greater stability and lipase reactivity than
the two-carbon (C2) acetoxymethyl ether-masked substrates. We used
these new C4 and C8 probes to profile esterases and lipases from <i>Mtb</i>. The C8-masked probes revealed a new esterase band in
gel-resolved <i>Mtb</i> lysates that was not present in
lysates from nonpathogenic <i>M. bovis</i> (bacillus Calmette-Guérin),
a close genetic relative. We identified this <i>Mtb</i>-specific
enzyme as the secreted esterase Culp1 (Rv1984c). Our C4- and C8-masked
probes also produced distinct <i>Mtb</i> banding patterns
in lysates from <i>Mtb</i>-infected macrophages, demonstrating
the potential of these probes for detecting <i>Mtb</i> esterases
that are active during infections
Total Syntheses of (±)-Securinine and (±)- Allosecurinine
Total syntheses of (±)-securinine and (±)-allosecurinine that employ a tandem rhodium carbenoid-initiated Claisen/α-ketol rearrangement sequence as a key step are described
An Optimized Enzyme-Nucleobase Pair Enables <i>In Vivo</i> RNA Metabolic Labeling with Improved Cell-Specificity
Current transcriptome-wide analyses have identified a
growing number
of regulatory RNA with expression that is characterized in a cell-type-specific
manner. Herein, we describe RNA metabolic labeling with improved cell-specificity
utilizing the in vivo expression of an optimized
uracil phosphoribosyltransferase (UPRT) enzyme. We demonstrate improved
selectivity for metabolic incorporation of a modified nucleobase (5-vinyuracil)
into nascent RNA, using a battery of tests. The selective incorporation
of vinyl-U residues was demonstrated in 3xUPRT LM2 cells through validation
with dot blot, qPCR, LC-MS/MS and microscopy analysis. We also report
using this approach in a metastatic human breast cancer mouse model
for profiling cell-specific nascent RNA