C-LANA FP assay Measured in Biochemical System Using Plate Reader - 2117-01_Inhibitor_Dose_DryPowder_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: 16
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. 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
7. D505fp/D535 dichroic mirror
8. Apply fluorescence polarization formula to values and analyze data
PRESENCE OF CONTROLS: Neutral control wells (NC; n=132) and positive control wells (PC; n=18) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal (10)(Max_Concentration).
The raw signals of the plate wells were normalized using the 'Compounds' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate sample compound wells (SC) is set to a normalized activity value of 0.
A normalized activity value of 100 is defined as (2)(SC).
A normalized activity value of -50 is defined as (0.5)(SC).
Experimental wells values were scaled to this range.
PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied.
MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): AC50
AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
AC values were calculated up to the active concentration limit described for each sample.
pAC was set to equal -1*log10(AC)
Activity_Outcome = 1 (inactive) when:
a) compound shows activity but in a direction opposite to the expected outcome
in these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to null
b) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
c) AC > AC_limit
Activity_Outcome = 2 (active) when:
AC <= AC_limit
Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= -70% but <= -30%, or
b) The fit was deemed not valid due to poor fit quality.
If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),
then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
When the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.
When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.
The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.
All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.
Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)