Optimisation of FRET parameters to improve FRET compound screening assay, viz 1. Denaturation regimen 2. Establishing Km of [ATP] substrate

1. Achim established some basic parameters, i.e.Achim established some basic parameters, i.e.
a) Optimal combination of fluorophores with Ref. to REL 1
b) Optimal fluorophore concentrations
c) Optimal ratios of fluorophores with bridge
d) Efficacy of Baculovirus REL 1 with respect to above set up
My contribution thus far:My contribution thus far:
e) Compared activities of E Coli expressed R1-A2 with aforementioned Bac
prep
f) Looked at potential stabilising effect of 0.1 % Triton X-100
g) Investigated sensitivity of E Coli R1-A2 (in context of assay) to various
conc. of DMSO
h) Looked at effect of Ligation time on signal magnitude
i) Investigated different denaturation regimens
j) Preliminary Investigation of [ATP] on signal output:
k) Establishment of Vmax and Km
l) Batch analysis to establish quality index or Z score

2. | | | | | | | | | | | | | | | | | | | | | | | | |
D
hγ2hγ1
Cy5Cy5FAMFAM
gR#1gR#1
5’ #15’ #1
3’ #23’ #2

3. Denature & anneal 1uM 5’ # 2 + 3’ #1 + gRNA #1 @ 72o
C 2 min followed by
Slow cooling (0.1o
C/sec) to 20o
C ( in presence of 40uM ATP + 5mM MgCl2 )
Subsequently ligate single labelled species for 30 min to 1 hour @ 27o
C using
(100ng) of E Coli R1-A2 (#001_6_x) in presence of 0.1% Triton X-100 & 10 x adenylation
buffer + 40uM ATP
Terminate reactions by adding EDTA + gDNA (complementary to g R#1) & heating
@ 95o
C for 2 min
Quantify FRET emission @ 670nM using Micro plate reader
| | | | | | | | | | | | | | | | | | | | | | | | |
D
A
D
hγ2hγ1
D hγ2
hγ1
LigationLigation
No LigationNo Ligation
Denature
gR #1
5’ #2
3’ #1

4. Specific Signal Versus L1-A2 Batch (#001)
#001_1
#001_2
#001_3
#001_4
#001_5
#001_6
#001_6_6/7
#001_6_8
#001_6_9
#001_6_10
T4RNALigase2
No L1
0.0
10000.0
20000.0
30000.0
40000.0
50000.0
60000.0
#001_1-6 + T4 RNA ligase 2 + No L1 control
Batch #_#001
AFU

5. The effect of DMSO on FRET Ligation: #001- 6_5/2 Preparation (100ng per reaction)
0.0
20.0
40.0
60.0
80.0
100.0
120.0
140.0
0.00% 0.20% 0.40% 0.60% 0.80% 1.00% 2.00% 5.00% 10.00%
% DMSO in ligation mix (ligation time = 30 min)
SpecificSignal(%of'no'DMSO)

6. FRET Emission versus ligation time versus REL 1 Conc
0.0
10000.0
20000.0
30000.0
40000.0
50000.0
60000.0
70000.0
0 min 1 min 5 min 10 min 20 min 40 min 60 min 90 min 120
min
Time of Ligation
SpecificFluorescentEmission
222.5ng #001_6_3/4
22.25ng #001_6_3
111.25ng #001_6_3/4

7. FRET Emission versus Ligation time: The effect of 0.1% Triton X-100 on resultant signal
0.0
10000.0
20000.0
30000.0
40000.0
50000.0
60000.0
70000.0
0 min 1 min 5 min 10 min 20 min 40 min 60 min 90 min 120 min
Ligation time
SpecificFluorescence
111.25ng #001_6 + 0.1% Triton X-100
111.25ng #001_6; No TX-100
No Enzyme control
111.25ng #001_6 + 0.1 % TX-100 + 1ug/ul BSA

8. 111.25ng +0.1% TX-100111.25ng +0.1% TX-100
222.5ng. No TX-100222.5ng. No TX-100
111.25ng. No TX-100111.25ng. No TX-100
22.5ng. No TX-10022.5ng. No TX-100
No Enzyme
Control_#003
No Enzyme
Control_#004
FRET Emission versus Ligation time: The effect of REL 1 mass +/- 0.1% Triton X-100 on resultant
signal
0.0E+00
1.0E+04
2.0E+04
3.0E+04
4.0E+04
5.0E+04
6.0E+04
7.0E+04
0 min 1 min 5 min 10 min 20 min 40 min 60 min 90 min 120 min
Ligation time
SpecificFluorescence
111.25ng #001_6 + 0.1% Triton X-100
111.25ng #001_6; No TX-100
No Enzyme control
111.25ng #001_6 + 0.1 % TX-100 + 1ug/ul BSA
222.5ng #001_6
22.5ng #001_6
No Enzyme control_#003
# 003

9. The effect of denaturation regimen +/- g R#1 Annealing
gR#1_noheat
gR#1_noheat
gR#1_noheat
gR#1_noheat
gR#1+heat
gR#1+heat
gR#1+heat
gR#1+heat
NogR#1_noheat
NogR#1_noheat
NogR#1_noheat
NogR#1_noheat
NogR#1_heat
NogR#1_heat
NogR#1_heat
NogR#1_heat
0
5000
10000
15000
20000
25000
30000
#139_ No #139 80 % Form 80 % DMSO
Denaturation conditions
AFU
g R#1_no heat
gR#1 + heat
No gR#1_no heat
No gR#1_heat
In the presence of g R #1 irrespective of denaturation
consistently higher signal
In the absence of g R#1 signal +/- heat approx the same
In the absence of #139 heat denaturation less effective
For both formamide and (especially DMSO) significant signal
quenching apparent
This quenching was confirmed by similar magnitude of effect
associated with signal from double labelled oligo control
Optimal conditions in terms of S/B = + g R #1 + #139 = 4.5

11. Set up 45 x reactions with L1; 45 reactions without L1
Compute sample (µs) and control means (µC)
Compute sample (σS) and control (σC) std deviations
Now compute 99% confidence interval for each cohort, viz 3σ
Dynamic range of assay =Dynamic range of assay = µs - µµs - µCC Want this to be as large as possibleWant this to be as large as possible
Separation band of assay = (µs - µSeparation band of assay = (µs - µCC) – () – ( 3σS - 3σC)
Effective S:B
Large !
Effective data variance:
Small !
Z Factor = Separation band = (µs - µ(µs - µCC) – () – ( 3σS - 3σC)
Dynamic range (µs - µ(µs - µCC))
= 1 -1 - (( 33σσSS - 3- 3σσCC)) Thus, larger numbers = better assay !
(µs - µC)(µs - µC)

12. Assessing QC of HTS (FRET assay ) using 'Screening window Co efficient' (or Z factor)
0
10000
20000
30000
40000
50000
60000
0 10 20 30 40 50 60
Sample #
AFU_Individualdatapoints
Plus L1_#013
Plus L1_#012
Minus L1_#012
Minus L1_#013
#012 & #013
rS/B = Mean Signal = 8.6 & 10.5
Mean Background
rS/N = Mean Signal - Mean background = 131.6 & 265.9
Standard deviation of background
rZ Factor = 0.6 & 0.6: (1.0 > Z > 0.5) = 'Excellent'

13. Signal intensity versus Ligation time versus [ATP]
7000
12000
17000
22000
27000
32000
37000
0µM_ATP 1µM_ATP 10µM_ATP 100µM_ATP 200µM_ATP 500µM_ATP
[ATP]
AFU
10 min Ligation
20 min Ligation
40 min ligation
60 min ligation

14. From 1:2 serially diluted oligo @ specified concentration, select ‘spline’ on data
curve accommodating data range (AFU) of actual ligated FRET products
Calculate the slope of the Spline, i.e. change in AFU per unit of concentration =
‘concentration coefficient’
Using this data range calculate [ligated product] by dividing AFU [ligated product]
by the ‘concentration coefficient’
[Product] conc. estimated in this way is then used to compute initial velocityinitial velocity
((νν0)0) in nM/min by dividing product appreciation (nM) by 30 (min), i.e. increase from
10 to 40 mins of ligation, culminating in ν0 in units of nM/min
Finally, to compute VmaxVmax and hence KmKm (= [product ] @ Vmax /2) plot [product] on
y-axis against [ATP] on x-axis (= substrate conc.) and subject to non linear
regression analysis using rectangular hyperbolic model with GraphPad®

15. ΔΔ AFUAFU == Conc. CoefficientConc. Coefficient
ΔΔ [nM][nM]

16. Serial Diln Double labelled Oligo: Log2 AFU versus Oligo conc.
y = - 0.8679x + 16.63
R2
= 0.9913
8
9
10
11
12
13
14
15
16
100 50 25 12.5 6.25 3.125 1.5625 0.78125
[Oligo]_nM
Log(2)AFU
Log(2) Specific AFU
Linear (Log(2) Specific AFU)

19. AFU versus time for different [ATP]
5000
10000
15000
20000
25000
30000
35000
40000
10 min ligation 20 min ligation 40 min Ligation 60 min Ligation
Ligation time
AFU
0uM ATP
1uM ATP
10uM ATP
100uM ATP
200uM ATP
500uM ATP

20. Estimated (ligated) product Conc. (from Double labelled Oligo std curve) vs Substrate [ATP]
10
15
20
25
30
35
40
45
10 min 20 min 40 min 60 min
Ligation time
[product]nM
0uM ATP
1uM ATP
10uM ATP
100uM ATP
200uM ATP
500uM ATP

21. KKmm ~ 4~ 4µµMM
95% confidence interval =095% confidence interval =0µM-9µMµM-9µM
RR22
= 0.9360= 0.9360

22. Initial Velocity ( nM/min)
Michaelis-Menten
Best-fit values
Vmax 0.3726
Km 3.675
Std. Error
Vmax 0.02677
Km 1.877
95% Confidence Intervals
Vmax 0.2983 to 0.4469
Km 0.0 to 8.887
Goodness of Fit
Degrees of Freedom 4
R square 0.936
Absolute Sum of Squares 0.007559
Sy.x 0.04347
Constraints
Km Km > 0.0
Number of points
Analyzed 6

23. Repeat Km/Vmax assay but with a minimum of 8 [ATP] subtending Km
Thus, >/= 8 x [ATP] approximating to 0.2 x – 4.0 x Km :
 0µM
 1µM
 2µM
 4µM
 6µM
 8µM
 10µM
 20µM
 30µM
 50µM
 100µM
Firstly however determine
 Optimal [Mg]Optimal [Mg] in context of (annealing &/or ligation) adenylation buffer:
0.5mM – 10mM (final)
 Tris versus HEPESTris versus HEPES in adenylation buffer
 pH optima of preferred buffer:pH optima of preferred buffer: 6.5; 7.0; 7.5; 8.0; 8.5
Also investigate in
Context of Enz diln.
buffer