C-LANA FP assay Measured in Biochemical System Using Plate Reader - 2117-01_Inhibitor_SinglePoint_HTS_Activity
C-terminal LANA self-associates to bind a specific 20 bp terminal repeat (TR) DNA sequence (Kd of 1.51+/- 0.16 nM) on the KSHV episome. LANA binding is essential for episome persistence since it tethers the KSHV DNA episome to host chromosomes and is essential for LANA to mediate replication of TR DNA. We developed a primary assay to detect the binding of LANA binding sequence (LBS) (a labeled more ..
BioActive Compounds: 262
Keywords: KSHV, LANA, DNA-binding, latency, fluorescence polarization
C-terminal LANA self-associates to bind a specific 20 bp terminal repeat (TR) DNA sequence (Kd of 1.51+/- 0.16 nM) on the KSHV episome. LANA binding is essential for episome persistence since it tethers the KSHV DNA episome to host chromosomes and is essential for LANA to mediate replication of TR DNA. We developed a primary assay to detect the binding of LANA binding sequence (LBS) (a labeled DNA duplex containing the sequence specific binding site) to MBP- fused C-terminal LANA (C-LANA, residues 932-1162) and assess the specificity of the inhibition. The primary assay uses fluorescence polarization to determine if LBS binds to its C- LANA binding partner after addition of compound. Using our primary assay, acridine orange has an IC50 less than 10 uM, showing that intercalation into DNA is sufficient to abrogate LANA binding to LBS. Although acridine orange inhibits C-LANA binding, and will be useful as a control, it is not an ideal probe since it non-specifically intercalates into DNA.
Polarization of emitted light is low for unbound oligonucleotide LBS tracer, but upon C-LANA binding, polarization increases. Compounds considered to be hits will inhibit the binding of C-LANA to LBS (i.e. inhibitory compounds will decrease polarization).
MBP C-LANA (MBP fused to C-LANA residues 932-1162), 85 nM final concentration
6-FAM labeled 20 bp DNA oligonucleotide duplex, 10 nM final concentration (aka LBS)
Control: Acridine orange, 4 uM final concentration
Buffer: 20 mM Tris-HCl pH=7.5, 200 mM NaCl, 1 mM DTT, 10 ug/mL BSA, 0.01% Triton X-100
6Fam 5' labeled strand: 5'-CGG CCC CAT GCC CGG GCG GGA-3'
Complementary strand (unlabeled) : 5'-TCC CGC CCG GGC ATG GGG CCG-3'
Buffer recipe for 1 liter:
20 mL 1M Tris-Cl, pH 7.5
80 mL 2.5M NaCl
154 mg DTT
10 mg BSA
1 mL 10% Triton-X-100
1.Remove 1536 well assay ready plate (ARP) from incubator, de-lid plate.
2.Add 4 uL 2x LBS to all wells of the 1536 well Aurora plate with Thermo Combi nL
3.Add 4 uL 2x MBP-C-LANA with Beckman Coulter's Bioraptr to all wells of the 1536 well black Aurora plate
4.Add 0.5 uL of positive control to designated wells with the Bioraptr
5.Re-lid plate, return plate to Liconic incubator and incubate at room temperature for 1 hour
6.Read fluorescence polarization with Perkin-Elmer Viewlux plate reader using 480nm excitation filter, 535nm S and P emission filters and D505fp/D535 dichroic mirror
7.Apply fluorescence polarization formula to values and analyze data
PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of 100.
Experimental wells values were scaled to this range.
PATTERN CORRECTION: The plate pattern correction algorithm 'Runwise Pattern (Multiplicative)' in Genedata (v7.0.3) was applied to the normalized plate data.
This was set as equal to the maximum of the normalized and corrected sample replicate activities, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at 60.
PERCENTAGE OF ACTIVE REPLICATES:
For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.
The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at 100.
Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_T
Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_T
Samples passing AT only were assigned an outcome of 2 (active) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T
tSamples passing PAR_T only were assigned an outcome of 2 (active) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)