click chemistry - jena bioscience · click chemistry on nucleic acids 1. definition and hallmarks...
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Jena, October 24th 2017
Click Chemistry –Expanding the Scope of Nucleic Acid Labeling
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Click Chemistry on Nucleic Acids
1. Definition and Hallmarks of a Click Reaction
2. Classification of Click Reactions
3. In vitro Synthesis of DNA and RNA Conjugates
4. Clickable Nucleosides in Metabolic Labeling
5. Trends and Perspectives
2
“Diverse Function from a Few Good Reactions“[1]
§ Selective: Formation of only the desired
product
§ Orthogonal: No side reaction with sample matrix
§ Simple: Easy to do for a non-chemist
§ Fast: Completion in biomolecular timescale
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K. Barry SharplessNoble Price Chemistry 2001
A + B A BClick?
[1] Kolb et al. (2001) Angew. Chem. Int. Ed. Engl. 40(11): 2004
[1] Rostovtsev et al. (2002) Angew. Chem. Int. Ed. Engl. 41(14):2596[2] Tornøe et al. (2002) J. Org. Chem. 67(9):3057[3] Agard et al. (2004) JACS. 126(46):15046
Azide Clicks with Alkyne in the Presence of Cu(I) or throughRing Strain
A B+Cu(I)
(CuAAC)
Alkyne-containingmolecule A
Azide-containingmolecule B
Conjugate of A and B, cross-linked via a Triazole moiety
AB
NNN
NNN
N
O
N
ONN
N
AB
Azide-containingmolecule B
NNN
DBCO-containingmolecule A
A
B+ SPAAC
N
NNN
N NN
NNN
HOO
N
NNN
N NN
NNN
HOHO
OHTHPTA BTTAA
• CuSO4, reduced in situ• Ascorbate
Cu(I)-stabilizingLigands
4
Tetrazines Click with Alkenes
A
Tetrazine-functionalizedmolecule A
+Vinyl-
functionalizedmolecule B
B
NN N
N
R2
R1
B
A
NNH
R2
R1
Conjugate of A and B crosslinkedvia Dihydropyrazine
B+
Methylcyclopropene-functionalized
molecule B
B+
trans-Cyclooctene(TCO)-
functionalizedmolecule B
B
A
NNH
R2
R1
A
BNNH
R2
R1
Carboni and Lindsey (1959) JACS 81(16):4342.5
How Fast is Fast Enough?
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A + B A Bk2
NHR
O
NN N
N
R1
R2
+N
NN
+
N
O
NNN
+
M-1s-1
Isolated Material Fixed Cells/Life Cell Surface Life Cells Cytosol
10-3 10-2 10 102 106
NH
O
ONO
OH
HON O
O
N O
O
+
NN N
N
R1
R2
+
NN N
N
R1
R2
[1] Oliveira et al. (2017) Chem. Soc. Rev. 46(16):4895[2] Kozma et al. (2017) ChemBioChem. 18(6):486
Slow Fast
Click Chemistry on Nucleic Acids
1. Definition and Hallmarks of a Click Reaction
2. Classification of Click Reactions
3. In vitro Synthesis of DNA and RNA Conjugates
4. Clickable Nucleosides in Metabolic Labeling
5. Trends and Perspectives
7
Dual DNA Labeling for FRET and TIRF in One Pot
§ CuAAC (and SPAAC) are orthogonal to iEDDA
§ Single step/ one pot procedure for DNA labeling
§ è Easy combination of diverse labels
§ è No de novo synthesis of DNA required
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CuSO4,THPTA
Ascorbate
[1] Schoch et al. (2012) Bioconjug. Chem. 23(7):1382
Polymerases:Pwo,Vent (exo-)KOD XL
Internal Modification of Long DNA by Incorporation ofFunctionalized dNTPs
§ Tolerance of most DNA-polymerases towards modified dNTPs
§ C-5-modification of dU/dC particularly well suited (major groove)
§ Variable degree of substitution by adjusting dNTP/dNTP*
§ Modified amplicons of several kb accessible
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[1] Burley et al. (2006) JACS 128 (5):1398[2] Gierlich et al. (2007) Chem. - Eur. J. 13 (34):9486
NH 3
2NH1
6
5 4
O
7O
8
Efficient Synthesis and KOD XL- Incorporation of 5-N3-dCTP
10[1] Krause et al. (2014) Chem. - Eur. J. 20(50): 16613
Preparing RNAs beyond the Scope of Solid Phase Synthesis
§ C5-modified UTP derivatives are substrates of the T7-RNA polymerase
§ Generally, fewer polymerases available than for DNA/PCR
§ Synthesis of several kb transcripts possible in vitro
§ Constraints apply due to conserved promoter sequences
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[1] Rao et al. (2012) Chem. Commun. (Camb). 48(4):498[2] Sawant et al. (2016) Nucleic Acids Res. 44(2):e16
Sawant et al. (2016) Nucleic Acids Res. 44(2):e16
NH
O
ONO
OHOH
OPOOH
OPOPHOOO
OHOH
N3
Chemo-Enzymatic 3′- and Internal RNA Modification UsingPolyA-polymerases and Ligases
§ Limited accessibility of long internally modified RNA by chemical means
§ Chemo-enzymatic approaches to overcome synthetic limitation
§ Azides are small
12[1] Winz et al. (2012) Nucleic Acids Res. 40(10):e78
Chemo-Enzymatic 3′- and Internal RNA Modification UsingPolyA-polymerases and Ligases
13[1] Winz et al. (2012) Nucleic Acids Res. 40(10):e78
Partially Complementary DNA Scaffolds Increase Click Yields
§ Increasing the efficiency of CuAAC on internal azide
§ Forcing the ligation site into a bulge
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75-85% without scaffold92% with scaffold
[1] Winz et al. (2012) Nucleic Acids Res. 40(10):e78
TdT permits 3′- and Internal Modification of DNA byTailing and Extension
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§ Transfer of various base-modified (d)NTPs by
terminal deoxynucleotidyl transferase TdT
§ Fill-up by polymerase or enzymatic ligation
[1] Winz et al. (2015) Nucleic Acids Res. 43(17): e110
T7 RNAP
SP6 RNAP
T3 RNAP
5-(d)UpG Dinucleotides Are Excellent Transcriptional Initiators
§ (d)UpGs are universally applicable
16[1] Samanta et al. (2014) Chem Commun. 50(11):1313
dUpG-Priming Allows to Decorate RNA with Metal Complexes, Affinity Tags, Sugars, Surfactants, Dyes, …
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A
B
C
[1] Samanta et al. (2014) Chem Commun. 50(11):1313
The 5′-OdUpG Label is a Ligation Donor for Internalization
§ Requirements of ligation approach:
§ Acceptance by polynucleotide kinase
§ Acceptance by ligases
§ 5-UpG modification tolerated bythree different enzyme classes
§ Two-step procedure
18[1] Samanta et al. (2014) Chem Commun. 50(11):1313
Chemo-Enzymatic Procedures Are Applicable to Any Positionin Any Nucleic Acid
InternalRandom Site-Specific
3′ 5′
DNA
RNA
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Click Chemistry on Nucleic Acids
1. Definition and Hallmarks of a Click Reaction
2. Classification of Click Reactions
3. In vitro Synthesis of DNA and RNA Conjugates
4. Clickable Nucleosides in Metabolic Labeling
5. Trends and Perspectives
20
Cytoplasm
Nucleus
Base Base Phosphorylation ofnucleoside analog by
cellular kinases
In vivo Application of Clickable Nucleosides: Metabolic Labels
DNAP
A Replication
RNAP
B Transcription
PAP
C Polyadenylation
NH
O
ONO
OH
HO
NH
O
ONO
OH
HO
NH
O
ONO
OH
HO
N3N
NH2
ONO
OH
HO
NH
O
ONO
OH
HO F
[1] Salic and Mitchison (2008) PNAS 105 (7):2415[2] Neef and Luedtke (2011) PNAS 108(51): 20404[3] Guan et al. (2011) ChemBioChem 2 (14):2184[4] Neef and Luedtke (2011) Chembiochem. 15(6):789[5] Rieder and Luedtke (2014) Angew. Chem. Int. Ed. Engl. 53(35):9168[6] Jao and Salic (2008) PNAS 105(41): 15779[7] Curanovic et al. (2013) Nat. Chem. Biol. 9 (11):671 21
Click Chemistry Permits Direct Introduction ofAffinity Tags
§ Analysis of mRNA-polyadenylation
§ Biotin tag ideally suited for streptavidin pulldown and NGS22
Feeding
N
NN
NNH2
O
OHOH
HO
Lysis/ RNA-isolation
CuAAC
Biotin-N3N
NN
S
NHHN
O
Agarose GelSeparation
Northern-Blot
Strep-AP
LuminescenceDetection
Curanovic et al. (2013) Nat. Chem. Biol. 9 (11):671
Superior Efficiency of Click-Labeling to Immunostaining
§ No Antibodies required
§ Label can easily be adapted to individual needs
§ Mutual orthogonality of both techniques23
FixationFeeding
NH
O
ONO
OH
HO
Br
Yanti-Br
BrBrBr
BrBrBr
BrBrBr
Yanti-Fc
BrBrBr
Y
A
NH
O
ONO
OH
HO
N3
FixationFeeding Click
B
Visualization of Metabolically Labeled DNA and RNA
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Salic and Mitchison (2008) PNAS 105 (7):2415
NH
O
ONO
OH
HO
Rieder and Luedtke (2014) Angew. Chem. Int. Ed. Engl. 53(35): 9168
NH
O
ONO
OH
HO F
NH
O
ONO
OH
HO
NH
O
ONO
OH
HO
Br
Jao and Salic (2008) PNAS 105(41): 15779
NH
O
ONO
OHOH
HO
Click Chemistry on Nucleic Acids
1. Definition and Hallmarks of a Click Reaction
2. Classification of Click Reactions
3. In vitro Synthesis of DNA and RNA Conjugates
4. Clickable Nucleosides in Metabolic Labeling
5. Trends and Perspectives
25
The (Last) Challenge Remaining- Life Cell Imaging
§ Already well established in protein science
§ Metabolic nucleotide labels are rather slow
§ Cytotoxicity of copper
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[1] Carlson et al. (2013) Angew. Chem. Int. Ed., 52(27): 6917[2] Uttamapinant et al. (2012). Angew. Chem. Int. Ed. Engl. 51 (24):5852.
N B- N+
FF
NN
NN
R
+N B- N
+
FF
NHN
R
OHOH
1600 x turn-on
Turn-On-Tetrazines[1]
N
O
NH
NN N
Cu+
R+N
O
NH N
NNR
10 x decreasedCu(I)-demand
Picolyl Azides[2]
Click Chemistry Is Useful for Anybody Dealing withNucleic Acid Analytics
§ Click reactions are broadly applicable to nucleic acids
§ High modularity in experimental design
§ Small tags are well tolerated by various enzyme classes
§ Allow preparation of previously inaccessible conjugates
§ Virtually any click precursor commercially available
§ Life cell applications subject to current research
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