Use of a biochip technology for the measurement of glycosylase activities Sylvie Sauvaigo, Valérie Guerniou, Didier Gasparutto and Jean-François Millau Laboratoire « Lésions des Acides Nucléiques », DRFMC/SCIB-UMR 5046 CEA Grenoble, 17 rue des martyrs 38054 Grenoble Cedex 9 France Research purpose
[email protected] Repair of DNA damage involves a very complex network of interacting proteins. Glycosylases are in charge of eliminating double-helix non distorting DNA lesions. Determination of the substrate specificity for each known prokaryotic or eukaryotic enzyme as well as precise measurement of glycosylase activities within either purified preparations or crude cell extracts constitute the matter of more and more publications in the literature. Studies are usually performed with radio-labeled synthetic oligonucleotides containing targeted lesions. The digestion products are analyzed after electrophoresis on denaturing polyacrylamide gels. It seemed then interesting to set up a versatile and generic tool that could help to study the repair of different lesions at the same time, to get rid of the radio-labeled material, and sensitive enough to be used for clinical evaluation of the role of DNA damage repair. We thus designed a low density microchip tool functionnalized with different lesions bearing oligonucleotides (1). OLIS A : microwells functionnalization configuration 1 2
12
Principle of the microwells functionnalization (indirect modified oligonucleot ide fixation)
10 µM oligonucleotide spots- chemical fixation by 5 ’ e nd- Indire ct hybridization with modifie d oligonucle otides
A
B IO
-TTTTT-CCA CAC GGT TAT CAG TC Oligonucleot ides -TTTTT-CCA CAC GGT TAT CAG TC -TTTTT-CCA CAC GGT TAT CAG TC -TTTTT-CCA CAC GGT TAT CAG TC
S pott ed s ingle s trand signal control O
Principle of glycos ylas e dete ction on biochips
linked t o the s upport Uns table hybrid
S table hybrid
HN
H
K
uracil
O
N H HN
H
N O
N
N
B IO
M
N
J
inosin
I
A/ 8 -oxo-G ua
N
K
L
B IO
I
GGT GTG CCA TCC ATA GTC AG CTT GAT CAC CTA GGG GGC CCG ACG 3’ 5’
N
M
F (formylamine)
N
et heno-dA
O
J
N N H
N
Glycos ylas e activity
S ignal los s
les ion Biotin + S treptavidin-Phycoerythrin
3’ 5’ O -TTTTT-CCA CAC GGT AGG TAT CAG TC GAA CTA GTG AT CCC CCG GGC TGC----BIOTIN GGT GTG CCA TCC ATA GTC AG CTT GAT CAC CTA GGG GGC CCG ACG 3’
H
H
N
H J
I
NH
H
H
L
S ignal
3’ 5’ O GAA CTA GTG AT CCC CCG GGC TGC----BIOTIN
NH2
N
H
H
K
M
NH
O
8 -oxo-G ua/ C
B IO
Modifie d oligo
5’ 3’ -TTTTT-CCA CAC GGT AGG TAT CAG TC
O
5’
N
Junction oligo
N
We used commercial (OLIgo Sorbent Array-APIBIO, Grenoble, France) microchips constituted of microwells functionnalized with different oligonucleotides to set up the appropriate hybrialization and enzymatic digestion conditions. Each well comprises 17 distinct spots including spotting and revelation controls. The lesion bearing oligonucleotides, that are also labeled by a 3 ’biotin, were indirectly hybridized. After incubation with the glycosylase containing medium and washings, the modified oligonucleotides remaining on the support were revealed. The revelation was done by incubation with streptavidin-phycoerythrin and the different signals were integrated using an appropriate software (ApiAnalyser-APIBIO) after fluorescence microscope image analysis. Materials and methods The reaction volume in each well is 35 µl. The oligonucleotides (1pmole of each) are hybridized all together in two steps for 1h at 37°C in PBS containing 0.2M NaCl (first step: junction oligos; second step: modified oligos). Each well is then incubated with the enzyme for indicated time at 37°C. The wells are washed for 30 min in PBS/NaCl 0.2M/tween 20 0.05% at 37°C. Hybridization between junction oligos and modified oligos may also be performed separatly in a test tube before fixation of the hybrid within the well. The wells are then incubated for 1h at 37°C with strept-PE. The microscope analysis is then immediatly performed. All the signals of one well are integrated at the same time for 1,5 s. The system automaticaly proceeds to the reading of the whole plate. Some preliminary experiments were performed using extracts from HeLa cells prepared from the protocol of Collins et al. (2).
Results treatment The signals of the non digested oligos at time 0 were set up to 100. The amount of non digested oligos remaining on the support after enzymatic digestion was calculated as a percentage of these controls.The signals were normalized when necessary relatively to the values of the spotting and revelation controls. Examples of results Fpg digestion (all the oligonucleotides have different sequences)
Study of oligo sequence effect on digestion Fpg digestion (0,8 µg/ml)-Sequence effect
Fpg digestion (0,08 µg/well)
120
Signal evolution
Fpg digestion (0,8 µg/well) 140
120 control1
100
120
uracil
80
100
8oxo/dC
60
dA/8oxo
40
inosin
80
ethenodA
0 20
40
60
8oxo1
40
8oxo2
20 20
40
60
80
Digestion time (min)
20 0
80
Control
60
0
40 0
0
80
0
60
formylamine
20
100
10
20
30
40
50
60
8oxo1= GAA CTA GTG OAT CCC CCG GGC TGC 3 ’ 8oxo2= GAA CTA GTG GAT CCC CCO GGC TGC 3 ’
70
Digestion by HeLa extracts (dU, CdU, HmdU, T and dF have the same sequence) Digestion by HeLa cellular extracts (1/20)
Digestion by HeLa cellular extracts (1/50) 140
120 CdU
60
dU
40
HmdU T
20
dF
0 0
20
40 time (min)
60
80
FordU
%
%
80
Bio1-3
120 100
Bio1-3
100
CdU
80
dU
60
HmdU
40 20
T dF
0
Conclusion A lot of impovements have to be brought to this assay (choice of oligo sequence, signal integration and quantification, extract digestion, controls…). However this tool is very powerfull and its use results in a significant gain of time and facilitates the study of lesion repair.
FordU 0
20
40
60
80
time (min)
(Bio=spotted single stranded controls, T=duplex control, CdU= 5-carboxyuracil, HmdU=5-(hydroxymethyl)uracil, FordU=5-formyluracil, dF=formylamine)
Bio2
References: (1) S. Sauvaigo. Patent WO0190408A (2) Collins et al. (2001) Mutagenesis. 16, 297-301.
