3-nitro-tyrosine as an internal quencher of autofluorescence enhances the compatibility of fluorescence based screening of OBOC combinatorial libraries.
In the one-bead-one-compound (OBOC) combinatorial method, compounds are constructed on bead resin via split-mix library synthesis such that multiple copies of the same compound are displayed on each bead. These libraries are rapidly screened with enzyme-linked colorimetric, fluorescent, radiometric, or whole-cell binding assays. While fluorescence-based probes are powerful tools in OBOC screening, their utility is greatly limited by the intrinsic fluorescence of many commonly used solid supports, (e.g. TentaGel), residual coupling reagents, and library compounds. To overcome this problem, we topologically partitioned TentaGel resin with a thin Fmoc-protected outer layer and an unprotected inner core. The inner core was derivatized with 3-nitro-tyrosine, followed by random peptide library construction. Spectral scans from a confocal microscope showed a dramatic decrease in the autofluorescence of blank beads and OBOC peptide libraries across a broad range of the optical spectrum. The quenching capacity of 3-nitro-tyrosine was also visualized in fluorescent micrographs. Using biotin/streptavidin as a model ligand/receptor system, we demonstrated a marked increase in visibility of three commercially available fluorescent probes binding to quenched beads, and increased feasibility of using a robust and efficient fluorescence-based, bead sorting platform known as COPAS. These data show that using 3-nitro-tyrosine as an internal quencher greatly enhances the compatibility of fluorescence-based applications and OBOC combinatorial screening.
Division of Hematology & Oncology, Department of Internal Medicine, UC Davis Cancer Center, University of California Davis, California, USA.
Jared
J
Townsend
Andrew
A
Do
Alan
A
Lehman
Seth
S
Dixon
Babak
B
Sanii
Kit S
KS
Lam
D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D005453:Fluorescence; D057166:High-Throughput Screening Assays / Q000379:methods*; D019151:Peptide Library; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry
D019151:Peptide Library; C002744:3-nitrotyrosine; D014443:Tyrosine