optimising HT FRET drug screening assay with ref to denaturation regimen; potentiation of soluble REL 1 production by heat shock inclusion

1.  Repeat Km evaluationRepeat Km evaluation
ImmediatelyImmediately
Near futureNear future
 Titrate K+ and Mg 2+ ions in 10 x Adenylation (ligation) bufferTitrate K+ and Mg 2+ ions in 10 x Adenylation (ligation) buffer
 Substitute Tris for Hepes (@pH 7.9) in bufferSubstitute Tris for Hepes (@pH 7.9) in buffer
 SubstituteSubstitute ββ ME for DTTME for DTT
 Upon completion repeat Z’ factor assay with reference to intra and interUpon completion repeat Z’ factor assay with reference to intra and inter
plate variabilityplate variability
Actually….Actually….
 Further investigated chemical denaturation conditionsFurther investigated chemical denaturation conditions
 Revisited L1 time curve kinetics to verify current #001 prep activity/kineticsRevisited L1 time curve kinetics to verify current #001 prep activity/kinetics
 Continued to optimise induction conditions as a prelude to a 2Continued to optimise induction conditions as a prelude to a 2ndnd
roundround
(#002) of L1 purification by affinity (His tag) LC(#002) of L1 purification by affinity (His tag) LC
Anticipated….Anticipated….

2. 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
S/B ~ 8 XS/B ~ 8 X
Assay # 008: 001-L1 ~ 3 months -80Assay # 008: 001-L1 ~ 3 months -80oo
CC
Gain =47Gain =47

3. Comparing FRET output from ligated products versus [ ATP ] versus L1 mass/rxn
NoL1
100ng
250ng
500ng
NoL1_5uMATP
100ng
250ng
500ng
0.0E+00
5.0E+03
1.0E+04
1.5E+04
2.0E+04
2.5E+04
3.0E+04
#2 + #1 No L1
AFU
3.1x 5.4x 6.0x
5µM
ATP
5µM
ATP
5µM
ATP
40µM
ATP
40µM
ATP
40µM
ATP
Fresh #001-6
3 month #001-6
Ligation less efficient with lower [ATP]Ligation less efficient with lower [ATP]
Consequently specific signal lessConsequently specific signal less
However, because background is exceptionally low, S/B – the real hall mark of assay quality - isHowever, because background is exceptionally low, S/B – the real hall mark of assay quality - is
actually higher ?!! Why ? Is the use of fresh DEPC water versus ‘old’ Sigma water at least part ofactually higher ?!! Why ? Is the use of fresh DEPC water versus ‘old’ Sigma water at least part of
the answer ?the answer ?

4. No significant appreciation in L1 activity over time course duration suggesting a loss of intrinsic activity
This precludes linear order reactions and inhibition studies
Thus new L1 must now be purified !!

5. Time course analysis of 3 L1 (#001) preps @ 111.25ng & 222.5ng versus T4 RNA ligase 2 (#033)
versus Assay #004B
T4 RNA Ligase 2,
40000.25T4 RNA Ligase 2,
37506.3
No L1,
2328.75
No L1,
2467.75
1ug/ul BSA
1ug/ul BSA
0
10000
20000
30000
40000
50000
60000
70000
0min 10min 20min 40min 60min 120min
Ligation time
AFU
#001-2-1_211010_111.25ng
#001-2-1_211010_222.5ng
#001-6_141010_111.25ng
#001-6_141010_222.5ng
#001-6-8_280610(dil)_112.5ng
#001-6-8(dil)_280610_222.5ng
T4 RNA Ligase 2
No L1
#001-6_2/5_Assay #004B_111.25ng
#001-6-2/5_#004B_1ug/ul BSA
Gain = 51
Blue = Fresh:
6 months @ -80o
C
Red = 1 week @
-20o
C
Violet = 4 months
(prediluted) @ -
20o
C
#033
2 months @ -80o
C
#004B
#033
L1 Now 6 months
No significant appreciation in L1 activity over time course duration (#033) suggesting a loss of intrinsic
activity
This pertains to all preps tested (~ 6 months old) irrespective of storage conditions
Curiously, the prep exhibiting least activity was newly thawed, i.e. ~ 6 months @ -80o
C ??!!

6. #1#1 #2#2 #3#3 #4#4
50KD
40KD
30KD
20KD
PP PP PP P?P?SS PP PP PP P?P?SS SS SS SS SS S?S? S?S?
+ + + +- - - -
Catalytic domainCatalytic domain
REL1REL1
Specific and high level expression of L1 (catalytic REL 1 domain) in pellet
No obvious sign of soluble protein !
Is this due to a lack of A2 (N-terminal TEV His tag) rendering L1 soluble ?
+/+/-- 0.5mM IPTG0.5mM IPTGWestern blotting
with:
 α REL 1Ab
 α His tag Ab

7. ++ ++-- -- ++ ++-- --
Bug buster/PBS + lysozyme/
Benzonase + 1% Triton X-100+ 1% Triton X-100
Buffer A + lysozyme/
Benzonase
+/- 0.5mM
IPTG
Buffer A + lysozyme/
Benzonase + 1% Triton+ 1% Triton
X-100X-100
Bug buster/PBS + lysozyme/
Benzonase
Clone #1_2Clone #1_2 Clone #5_1Clone #5_1
PPPP PP PP PPSS SS
Clone #1_3Clone #1_3 Clone #5_2Clone #5_2
SS SSPP PP PP PPSSSS SS SS SS
Cultures grown to an initial OD600 of 0.5_0.6 at 37o
C for 1.5 hours by seeding 2ml of LB with 25ul of overnight
Induction achieved by splitting 2ml culture into 2 x 1ml aliquots and then adding 6.25ul of 1/10 0.8M IPTG to one such
aliquot (2nd
= un induced control) followed by growth @18o
C for 20-21 hours
Like original prep (i.e. Initial OD600 0.8 followed by induction for 20 hours @ 20o
C in presence of 0.5mM IPTG) = #1_1
REL 1 remains in pelleted fraction
Similarly, TEV cleavable N terminal His tagged A2 remains associated with pellet
5050
4040
3030
2020
1515
1010
3.53.5
L1
51-469
A2
56-176
 4-12% Bis Tris Novex gel
 1 x MES
 200V 45 minutes
Pellet
#1_1 (+)
Supernatant
#1_1(+)

8. 50KD
40KD
30KD
20KD
PP PP PP PPSS PP PP PP PPSS SS SS SS SS SS SS
Catalytic domainCatalytic domain
REL1 (R1)REL1 (R1)
Chaperone (A2):Chaperone (A2):
N Terminal TEVN Terminal TEV
His TagHis Tag
#1#1 #2#2 #4#4 HS- #1HS- #1
+ + + +- - -- +/+/-- 0.5mM IPTG0.5mM IPTG
Heat shock @ 42o
C 10min; 37o
C 20 min; ice 30 min clearly solubilised some L1

9. 50KD
40KD
30KD
20KD
PP PP PP P+P+
SS
P+P+ P+P+ P+P+ PP
S+S+ SS S+S+ SS S+S+ SS S+S+
Catalytic domainCatalytic domain
REL1 (R1)REL1 (R1)
Chaperone (A2):Chaperone (A2):
N Terminal TEVN Terminal TEV
His TagHis Tag
+ 0.1mM+ 0.1mM + 0.5mM+ 0.5mM + 2.0mM+ 2.0mM No IPTGNo IPTG
Key:Key: P = pellet P+ = + heat shock
S = Spt. S+ = + heat shock
Less IPTG might actually be more effective at rendering L1
more soluble
In common with induction #5 heat shock may further promote
this trend

10. 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
EDTA + #139EDTA + #139
5mM Mg; 25mM K; no #139 = 1 x Adenylation buffer5mM Mg; 25mM K; no #139 = 1 x Adenylation buffer
Gain = 41Gain = 41
Can 1 x adenylation buffer potentiate specific signal or is this an artifact of high [mg] &/or no #139/EDTA ?

11. Manifest quenching of dual labelled Oligo by Formamide Titrants
#139STOP
#139STOP
#139STOP
25%
25%
25%
50%
50%50%
60%
60%
60%
70%
70%
70%
80%
80%80%
90%
90%
90%
100%
100%
100%
0
5000
10000
15000
20000
25000
30000
35000
40000
FRET FAM Cy5
Signal Output
AFU
#139 STOP
25%
50%
60%
70%
80%
90%
100%
Formamide @ all [ ] appears to be quenching FRET signal relative to #139 stopFormamide @ all [ ] appears to be quenching FRET signal relative to #139 stop
This can be attributed to quencing of FAM as Cy5 output comparable irrespective of conditionsThis can be attributed to quencing of FAM as Cy5 output comparable irrespective of conditions

12. Manifest quenching of dual labelled Oligo by DMSO Titrants > 50%
#139STOP
#139STOP
#139STOP
25%
25%
25%
50%
50%
50%
60%
60%
60%
70%
70%
70%
80%
80%
80%
90%
90%
90%
100%
100%
100%
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
FRET FAM Cy5
Signal Output
AFU
#139 STOP
25%
50%
60%
70%
80%
90%
100%
Above 50% [ ] DMSO appears to be quenching FRET signal relative to #139 stopAbove 50% [ ] DMSO appears to be quenching FRET signal relative to #139 stop
Below 50% DMSO FRET signal comparable to #139 stopBelow 50% DMSO FRET signal comparable to #139 stop
HoweverHowever ‘apparent’ parity of FRET may in fact belie quenched FAM output coupled to enhanced‘apparent’ parity of FRET may in fact belie quenched FAM output coupled to enhanced
Cy5Cy5

13. #139 stop signal magnitude plus heat almost equivalent to scaffold without g R#1 implying#139 stop signal magnitude plus heat almost equivalent to scaffold without g R#1 implying
efficient heat denaturationefficient heat denaturation
In the absence of heat some denaturation apparent for 50% DMSOIn the absence of heat some denaturation apparent for 50% DMSO
Apparently, denaturation by heat + DMSO less efficient than for #139 stop + heatApparently, denaturation by heat + DMSO less efficient than for #139 stop + heat
Both in the presence and absence of heat, DMSO mixtures should have included (molar excess)Both in the presence and absence of heat, DMSO mixtures should have included (molar excess)
#139 to preclude renaturation#139 to preclude renaturation
Thus, denaturation in this context undermined (potentially) by absence of #139: Include next time !Thus, denaturation in this context undermined (potentially) by absence of #139: Include next time !
FRETFRET

14. Regarding FAM signal from intact (+ g R#1) scaffold, #139 + heat yielded a higher output than in theRegarding FAM signal from intact (+ g R#1) scaffold, #139 + heat yielded a higher output than in the
absence of heat; This cab be attributed to quenching of signal by FRET in the latterabsence of heat; This cab be attributed to quenching of signal by FRET in the latter
In the case of DMSO, signal yielded by intact (+g R#1) scaffold consistently lower than for scaffoldIn the case of DMSO, signal yielded by intact (+g R#1) scaffold consistently lower than for scaffold
+ #139 stop buffer: Is this due to FRET quenching augmented by chemical quenching of FAM+ #139 stop buffer: Is this due to FRET quenching augmented by chemical quenching of FAM
molecules ?molecules ?
In common with #139 stop quenching of FAM by FRET in the context of DMSO indicated by augmentedIn common with #139 stop quenching of FAM by FRET in the context of DMSO indicated by augmented
signal in the presence of heat and moreover absence of gR #1 where scaffold formation is precludedsignal in the presence of heat and moreover absence of gR #1 where scaffold formation is precluded
FAMFAM

15. Cy5Cy5
Regarding Cy 5 output, consistent signal enhancement caused by DMSO (compared to #139 stop)Regarding Cy 5 output, consistent signal enhancement caused by DMSO (compared to #139 stop)
irrespective of heat application or not; inclusion of g R#1 or notirrespective of heat application or not; inclusion of g R#1 or not
This suggests an intrinsic effect on the Cy5 dye molecule itself by DMSOThis suggests an intrinsic effect on the Cy5 dye molecule itself by DMSO

16. Signal potentiation by adenylation buffer can be attributed to Mg & HEPES respectivelySignal potentiation by adenylation buffer can be attributed to Mg & HEPES respectively
Thus, could stop supplemented buffer supplemented with extra HEPES boost S/B ?Thus, could stop supplemented buffer supplemented with extra HEPES boost S/B ?
Caveat:Caveat: Will this interfere with efficient denaturation ?Will this interfere with efficient denaturation ?

17. ExtraExtra Hepes buffer does not interfere with efficient denaturationHepes buffer does not interfere with efficient denaturation
However neither does it augment S/BHowever neither does it augment S/B
Not suprising !!
Omitting #139 & EDTA culminates in less efficient denaturationOmitting #139 & EDTA culminates in less efficient denaturation
0.1X0.1X 1X1X 1X1X
0.1X0.1X
- #139- #139

18. 384 versus 96 well

19. Signal from both molarities of adenylation bufferSignal from both molarities of adenylation buffer
much higher than Fam-Cy5 oligo diluted in watermuch higher than Fam-Cy5 oligo diluted in water
Signal from 0.1 x strength and 1 x strength theSignal from 0.1 x strength and 1 x strength the
samesame
How does this signal compare with black 96 wellHow does this signal compare with black 96 well
plates ?plates ?
What components of the buffer augment signalWhat components of the buffer augment signal
compared with water alone and could these becompared with water alone and could these be
manipulated (in ‘stop buffer’) to boost S/B ?manipulated (in ‘stop buffer’) to boost S/B ?

20. The effect of diluent on signal emanating from 1:2 Serial Titration of Fam-Cy5 Double labelled Oligo: 96 well White
Greiner plate
y = -0.9156x + 12.584
R
2
= 0.9979
y = -0.9414x + 16.776
R
2
= 0.9995
y = -0.8545x + 16.365
R
2
= 0.9971
0
2
4
6
8
10
12
14
16
18
500 250 125 62.5 31.25 15.625 7.8125 3.90625 1.953125
Conc FAM-Cy5 Oligo_nM
Log(2)Specificsignal
Water Diluent
0.1 x Adenylation buffer diluent
1 x Adenylation buffer diluent
Log 2 water trend line
Log 2 Trend line 1 x adenylation
Log 2 0.1 x Adenylation trend line
Red = water
Yellow = 0.1 x
Pink = 1 x

21. S/B determined by dividing specific signal byS/B determined by dividing specific signal by
buffer/water without dye labelled Oligobuffer/water without dye labelled Oligo
S/B mirror previous absolute signal resultsS/B mirror previous absolute signal results

22. Signal associated with white Greiner plates falls toSignal associated with white Greiner plates falls to
background with diminished oligo less rapidly than forbackground with diminished oligo less rapidly than for
black plates, i.e. white plate sensitivity higherblack plates, i.e. white plate sensitivity higher
S/B for black plates could not be computed on account ofS/B for black plates could not be computed on account of
high gain, consequent higher background and –ive wellshigh gain, consequent higher background and –ive wells
below this backgroundbelow this background
Assay [ oligo }: should gain be adjusted to this point
for meaningful S/B determination ?

23. Sensitivity of white Greiner plate muchSensitivity of white Greiner plate much
higher than black Greiner platehigher than black Greiner plate
Consequently, gain can be set much lowerConsequently, gain can be set much lower
Thus, negative well higher than blank wellThus, negative well higher than blank well
This enables meaningful Signal to backgroundThis enables meaningful Signal to background
to be computedto be computed
Sensitivity of black plates lessSensitivity of black plates less
Consequently, gain adjustmentConsequently, gain adjustment
results in back ground signal >results in back ground signal >
blank wellsblank wells
This precludes meaningful S/BThis precludes meaningful S/B
calculationcalculation
However, should gain be adjustedHowever, should gain be adjusted
to conc. range corresponding toto conc. range corresponding to
actual [ dye labelled oligo ] ?actual [ dye labelled oligo ] ?

24.  Make 2 litre induction of L1-A2 using 0.1mM IPTG + heat shockMake 2 litre induction of L1-A2 using 0.1mM IPTG + heat shock
 Freeze away samples in thin walled 0.2ml tubes +/- cryoprotectants, viz.Freeze away samples in thin walled 0.2ml tubes +/- cryoprotectants, viz.
L-arginine & sulfobetainesL-arginine & sulfobetaines
 Quantify Licor gel with heat shock/IPTG titration and rerun mass equivalentQuantify Licor gel with heat shock/IPTG titration and rerun mass equivalent
samples for verification/quantitation of soluble L1 by Western blottingsamples for verification/quantitation of soluble L1 by Western blotting
 Repeat 50% DMSO denaturation + #139 and evaluate in context of mild temp.Repeat 50% DMSO denaturation + #139 and evaluate in context of mild temp.
gradient (viz. RT-60gradient (viz. RT-60oo
C)C)
 In addition try denaturation with 6M urea & DMSO + 1.5M (deionised) glyoxalIn addition try denaturation with 6M urea & DMSO + 1.5M (deionised) glyoxal
Before xmasBefore xmas
Near futureNear future
 Titrate K+ and Mg 2+ ions in 10 x Adenylation (ligation) bufferTitrate K+ and Mg 2+ ions in 10 x Adenylation (ligation) buffer
 Substitute Tris for Hepes (@pH 7.9) in bufferSubstitute Tris for Hepes (@pH 7.9) in buffer
 SubstituteSubstitute ββ ME for DTTME for DTT
 Perform Triton X-100 titration in ligation reactions & compare withPerform Triton X-100 titration in ligation reactions & compare with Brij-35Brij-35®®
 Upon completion repeat Z’ factor assay with reference to intra and interUpon completion repeat Z’ factor assay with reference to intra and inter
plate variabilityplate variability
 Accurately determine Km [ATP] in context ofAccurately determine Km [ATP] in context of E ColiE Coli L1 prep !!L1 prep !!