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Bioconjugation TechniquesBioconjugation Techniques
Hanadi SleimanHanadi Sleiman
Department of Chemistry, McGill UniversityDepartment of Chemistry, McGill University
References:References:
•• ““Bioconjugate TechniquesBioconjugate Techniques””, Greg T. Hermanson, Academic, Greg T. Hermanson, AcademicPress, 1996Press, 1996
•• ““Principles of Fluorescence SpectroscopyPrinciples of Fluorescence Spectroscopy””, Joseph Lakowicz, , Joseph Lakowicz, Third Edition, Springer, 2006Third Edition, Springer, 2006
•• ““Current Protocols in Nucleic Acid ChemistryCurrent Protocols in Nucleic Acid Chemistry””, Wiley, e, Wiley, e--Book @Book @McGillMcGill
•• Molecular Probes (Invitrogen) Molecular Probes (Invitrogen) http://probes.invitrogen.com/http://probes.invitrogen.com/
•• Glen Research Glen Research http://www.glenres.com/http://www.glenres.com/
Attachment of Fluorescent Labels to Biological MoleculesAttachment of Fluorescent Labels to Biological Molecules
•• Biological Assays:Biological Assays: Detection and monitoring of a protein or nucleic acidDetection and monitoring of a protein or nucleic acidfinding the function of genes/proteins,monitoring the finding the function of genes/proteins,monitoring the progress of a disease, etc.progress of a disease, etc.
•• Cell Biology: Cell Biology: mechanistic studies of biological transformations in cellsmechanistic studies of biological transformations in cells
•• Genomics, Molecular Biology, Proteomics, Medical DiagnosticsGenomics, Molecular Biology, Proteomics, Medical Diagnostics
R-NH2 Lysine, otherreactive amines
R'RHN
O
O
R' H
NaBH3CN
imine formation
reduction
R'RHN
acylation
N
O
O
O
O
R'N
C
S
R'
isothiocyanate
NH-R'RHN
O
Thiourea
Bioconjugation of Common Functional GroupsBioconjugation of Common Functional Groups
R O
O(aspartic acid, glutamicacid, other reactivecarboxylic acids)
NC
N
NH
EDC
N C NH
NHO O
R
O
N NN N
CDI
O
R N N
N
O
O
HOSO3Na
Sulfo-NHS(EDC)
R'-OHO
R OR'
ester
R'-NH2
O
R NHR'
amide
R'-NHNH2
O
R NHNHR'
hydrazide
N
O
O
O
SO3NaO
R
Bioconjugation of Common Functional GroupsBioconjugation of Common Functional Groups
Protocol (eg, for EDC coupling):Protocol (eg, for EDC coupling):1.1. Dissolve the protein in buffer (no amines or carboxylate buffersDissolve the protein in buffer (no amines or carboxylate buffers))2.2. Dissolve the molecule to be coupled in same buffer at 10Dissolve the molecule to be coupled in same buffer at 10--fold molarfold molar
excess to proteinexcess to protein3.3. Add the molecule solution to the protein solutionAdd the molecule solution to the protein solution4.4. Add EDC (concentrated solution in water, freshly prepared) to thAdd EDC (concentrated solution in water, freshly prepared) to the protein e protein
and molecule; EDC should be 10and molecule; EDC should be 10--fold molar excessfold molar excess5.5. React 2 hours at RTReact 2 hours at RT6.6. Purify the conjugate by gel filtration or dialysisPurify the conjugate by gel filtration or dialysis
RSH (Cysteine, otherreactive thiols)
N
O
O
R'
maleimide
N
O
O
R'
RS
Michael add.product
R'
HN
alkylation
OI
RS
Thioether
O
NH R'
R'S
S N
pyridyldisulfide
R'S
SR
Disulfide
Cysteine/cystine buried inside protein, reduction may cause disrCysteine/cystine buried inside protein, reduction may cause disruptionuptionof protein structure. of protein structure.
Bioconjugation of Common Functional GroupsBioconjugation of Common Functional Groups
Creation of new groupsCreation of new groups
HN
O
SO
NH2OH HN
O
SH
NH2 NH SH
N O
O
SO
O
O
SATA
H2NS
SNH2
EDCC N
HS
SNH2
O
COOH CO
SR
NH2 NH
COOH
O
O
ONH
O
O
OH
COOH CO
NH2
EDC
H2NNH2
NH2 NH
C
O
H
N O
O
O
O
O
H
Hohsaka, Nature Methods, 2006, 923Hohsaka, Nature Methods, 2006, 923
SiteSite--Selective incorporation of fluorophores to study protein conformSelective incorporation of fluorophores to study protein conformationation
FRET pairFRET pair
HOO
O
B5' (coupling)O
O
DMTO B
PO
CNN
Phosphoramidite
O
O
DMTO B
PO
CN
OO
OH
B
99.5%
O
O
DMTO B
PO
CNO
OO
O
B I2 / H20
(oxidation)
H+
(deprotection)
DNADNASynthesisSynthesis
Nucleic Acid BiolabelingNucleic Acid Biolabeling
OHNNNNNNNNNN
3'
5' OHNNNNNNNNNN
55’’--aminoamino--DNADNA
MMT : monomethoxytritylMMT : monomethoxytritylT : tritylT : trityl
55’’--thiothio--DNADNA
SequenceSequence--labeled aminolabeled amino--DNADNA
33’’--amino DNAamino DNA
Nucleic Acid BiolabelingNucleic Acid Biolabeling
TAMRATAMRA
Cy3Cy3
FluoresceinFluorescein
Nucleic Acid BiolabelingNucleic Acid Biolabeling
Nucleic Acid Biolabeling Nucleic Acid Biolabeling ––enzymatic incorporationenzymatic incorporation
Glen ResearchGlen Research
BiotinylationBiotinylation
NHHN
S
O
OH
O
Biotin
•• Can be Can be conjugatedconjugated to different to different biomoleculesbiomolecules
•• Possesses one of the strongest Possesses one of the strongest binding interactions with binding interactions with avidin/streptavidinavidin/streptavidin ((KKdd ~~ 1010--15 15 M)M)
•• ContainsContains four identical binding four identical binding sites for interaction with biotinsites for interaction with biotin
avidin / streptavidinavidin / streptavidin
Biotin (VitaminBiotin (Vitamin--H):H):
The BiotinThe Biotin-- Streptavidin InteractionStreptavidin Interaction
HN NH
S
O
-O
O
BiotinBiotin
HN NH
S
O
O
O
N
O
ONaO3S
HN NH
S
O
HN
O
NH
O
N
O
O
amino reactiveamino reactive
thiol reactivethiol reactive
BiotinylationBiotinylation
streptavidin
BiomoleculeBiomoleculetagged with biotintagged with biotin
Organic fluorophoreOrganic fluorophoretagged with biotintagged with biotin fluorescencefluorescence
quenchedquenched
Mohamed Slim, H. F. Sleiman, Bioconjugate Chemistry, 2004, Mohamed Slim, H. F. Sleiman, Bioconjugate Chemistry, 2004, 1515, 949, 949
Wavelength
Luminescence Intensity
0
10000
20000
30000
40000
50000
60000
70000
500 550 600 650 700 750
4 N
N
N
N
NN
Ru
HN
S
NHHN
O
O
+2
4 + AVIDIN N
N
N
N
NN
Ru
HN
S
NHHN
O
O
+2
NN
N
N
NN
Ru
HN
O
(PF6--)2
+2S
NHHNO
Ruthenium Ruthenium BipyridineBipyridine as as ChromophoreChromophore
MLCTMLCT
Emission from triplet MLCTEmission from triplet MLCT
•• Long lifetime ~ 1 Long lifetime ~ 1 μμss•• Large Stokes ShiftLarge Stokes Shift•• Resistance to Resistance to photobleachingphotobleaching•• ElectrochemiluminescenceElectrochemiluminescence(generates light catalytically)(generates light catalytically)
NN
N
NN
NRu 2+
Chem. Rev. 2004, 104, 3003-3036
Bingzhi Chen, Kim Metera, Rachel Nassif, N. SankaranBingzhi Chen, Kim Metera, Rachel Nassif, N. Sankaran
O
N OO
O
NO O
O
O
NH
N
NN
N
NNRu
O
2PF6
Phn m
O
OO O
O
O
O
O
O
O
O
XO
NO O
O
O
NO
SN
NO
H
H
Y
MicelleMicelle~ 10,000 luminescent~ 10,000 luminescentcenterscenters
BiomoleculeBiomolecule
B. Chen, K. Metera, H. F. Sleiman, Macromolecules, 2005, 38, 1084-1090
B. Chen, H. F. Sleiman, Macromolecules, 2004, B. Chen, H. F. Sleiman, Macromolecules, 2004, 3737, 5866 , 5866
Oregon Oregon GreenGreen
Barton, et al, JACS 2006Barton, et al, JACS 2006
Fluorescence probe for DNA mismatchesFluorescence probe for DNA mismatches
yellowyellow
redred yellowyellow
Is labeling of biomolecules always necessary?Is labeling of biomolecules always necessary?
cationic, conjugatedcationic, conjugatedpolymerpolymer
Leclerc, M.; Ho, H. A.; Boissinot, M. WO 02/081735 A2, 2002.Gaylord, B. S.; Heeger A. J.; Bazan G. C. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 10954.
Cowpea mosaic virusCowpea mosaic virus
Cu(I)Cu(I)
Cu(I)Cu(I)
Finn, Sharpless, et al,Finn, Sharpless, et al, J. AM. CHEM. SOC. 2003, 125, 3192
““Click ChemistryClick Chemistry”” –– Labeling of virus at all 60 sitesLabeling of virus at all 60 sites
Bertozzi, et al, Nature, 2004, 430, 873 Bertozzi, et al, Nature, 2004, 430, 873
Staudinger Reaction:Staudinger Reaction: in vivoin vivo biological labelingbiological labeling
•• The biolabeling The biolabeling ““marketmarket”” is lucrative; is lucrative; ““kitskits”” available for most jobsavailable for most jobs
•• Beware of Beware of ““black boxblack box”” attitude; need to understand the chemistryattitude; need to understand the chemistry
•• Think outside the Think outside the ““Molecular ProbesMolecular Probes”” boxbox
•• New fluorescent labels: metal centers, quantum dots, conjugatNew fluorescent labels: metal centers, quantum dots, conjugatededpolymers, etc.polymers, etc.
•• New trends: New trends: in vivoin vivo labeling with biolabeling with bio--orthogonal chemistryorthogonal chemistry
Broad comments:Broad comments: