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Title: Novel SAM analogs for sequence-specific DNA labeling
Other Titles: Nieuwe SAM analoga voor het sequentie specifiek labelen van DNA
Authors: Vranken, Charlotte
Issue Date: 13-Oct-2016
Abstract: DNA, the master blueprint of life, was discovered as our genetic material
only about 70 years ago. Since then, it has been studied in great detail
and numerous tools have been developed to decode it. The focus of this
dissertation is a technique called “DNA mapping”, which delivers long
range sequence information by inserting and locating fluorescent tags at
specific sites on the DNA substrate. The success of this technique relies on
high labeling efficiencies and preservation of the structure and integrity of
the DNA under study.
The labeling of DNA is typically performed with the aid of
methyltransferases, enzymes that normally transfer a methyl group from
the natural cofactor S - adenosyl-l -methionine (SAM) onto a biomolecule.
When tolerated, certain transferases can utilize modified cofactors and
deliver distinct groups rather than the simplest alkyl group. Here, we focus
on the synthesis of SAM analogs in which the methyl group is replaced by an
extended linker with a reactive functional group, which after DNA labeling,
can be used to tag the DNA with a fluorescent label through further coupling
reactions with suitably functionalized fluorophores.
In Chapter 1 of this doctoral thesis, I provide an overview of the known
labeling methods that are used to label biomolecules using SAM-dependent
methyltransferases and the necessary SAM analogs. In addition, the most
significant applications of this labeling technique are briefly surveyed.
In Chapter 2, I describe the synthesis of different SAM analogs and the
evaluation of their DNA labeling capability using a biological screening assay.
The common syntheses of these molecules are notoriously challenging and
low yielding. We have therefore extensively explored alternative synthetic
pathways; however, none seem to completely overcome the critical
challenges. A few of the synthesized cofactors contain useful functional
groups, suitable for further coupling reactions. We have tested the DNA
labeling capability of a selected few, containing amine, azide, and alkyne
groups, which gave positive results and are thus good candidates for further
coupling reactions with suitably functionalized dyes.
In previous work, low coupling efficiencies (~ 40%) were observed for amine to
NHS-ester reactions and therefore, in Chapter 3, we investigated the use
of an improved approach relying on a bioorthogonal and highly efficient
copper catalyzed azide-alkyne cycloaddition reaction. We first enzymatically
modified the DNA substrate with a terminal alkyne using a SAM analog,
which was then clicked to an azide containing dye. This reaction showed to
be much more efficient (~ 70%) than the NHS coupling technique, although
some DNA damage was observed due to the use of a transition metal catalyst. This was partially circumvented for small, plasmid DNA with the aid
of coordinating ligands but remained a problem for larger DNA fragments.
Therefore, there is still a need for more biological compatible coupling
reactions.
In Chapter 4, distinct fluorophore coupling reactions are compared, with
the aim of increasing labeling efficiency, while maintaining DNA integrity.
Here, the amine to NHS activated ester condensation, as well as the coppercatalyzed
click reactions and a strain-promoted copper-free 1,3-dipolar
cycloaddition were compared. The copper free click reaction did not only
show to completely preserve the DNA structure, but also to be the most
efficient coupling reaction of all three.
Chapter 5 demonstrates the chemoenzymatic synthesis of a SAM analog,
containing an emissive, isomorphic nucleobase, which is also active in
DNA methylation reactions. The novel cofactor could also be generated
in situ and directly be used for methylating plasmid DNA, which can
overcome cofactor stability issues. Spectroscopic analysis showed that
the SAM analog and its demethylated product were fluorescently distinct,
thus potentially providing a tool to monitor methyl transfer reactions using
optical techniques.
During this graduate study, I have synthesized several new SAM analogs,
which contain functional groups suitable for enzymatic DNA tagging and
further fluorescent labeling. Both the efficiency of the process and its impact
on the structural integrity of the DNA were investigated, as both factors are
key to obtaining a useful and reliable DNA map. Future work in this area will
need to concentrate on identifying effective synthetic pathways for SAM
analogs containing functionalizable groups or built in fluorophores.
To conclude, fluorescence-based techniques have become essential in
modern science. Regardless of the amazing advances in instrumentation
and its sophistication, these tools ultimately rely on the availability of
bright fluorophores and on practical methods for incorporating them into
biomolecules. While numerous approaches have been advanced, harvesting
the efficiency, specificity and mild operating conditions of enzymes for
creating unnaturally-modified biomolecules has emerged as an appealing
modern direction. Indeed, recent years have seen incredible progress in
bringing together the capacity of enzymes with synthetic chemists’ creativity
and skillful art of preparing novel cofactors. This direction is not without
challenges, but when successful it can alter the course of science. Superresolution
optical DNA mapping appears to be one such direction, which is
on the verge of becoming a game-changing tool for genetic analysis.
DNA, the master blueprint of life, was discovered as our genetic material
only about 70 years ago. Since then, it has been studied in great detail
and numerous tools have been developed to decode it. The focus of this
dissertation is a technique called “DNA mapping”, which delivers long
range sequence information by inserting and locating fluorescent tags at
specific sites on the DNA substrate. The success of this technique relies on
high labeling efficiencies and preservation of the structure and integrity of
the DNA under study.
The labeling of DNA is typically performed with the aid of
methyltransferases, enzymes that normally transfer a methyl group from
the natural cofactor S - adenosyl-l -methionine (SAM) onto a biomolecule.
When tolerated, certain transferases can utilize modified cofactors and
deliver distinct groups rather than the simplest alkyl group. Here, we focus
on the synthesis of SAM analogs in which the methyl group is replaced by an
extended linker with a reactive functional group, which after DNA labeling,
can be used to tag the DNA with a fluorescent label through further coupling
reactions with suitably functionalized fluorophores.
In Chapter 1 of this doctoral thesis, I provide an overview of the known
labeling methods that are used to label biomolecules using SAM-dependent
methyltransferases and the necessary SAM analogs. In addition, the most
significant applications of this labeling technique are briefly surveyed.
In Chapter 2, I describe the synthesis of different SAM analogs and the
evaluation of their DNA labeling capability using a biological screening assay.
The common syntheses of these molecules are notoriously challenging and
low yielding. We have therefore extensively explored alternative synthetic
pathways; however, none seem to completely overcome the critical
challenges. A few of the synthesized cofactors contain useful functional
groups, suitable for further coupling reactions. We have tested the DNA
labeling capability of a selected few, containing amine, azide, and alkyne
groups, which gave positive results and are thus good candidates for further
coupling reactions with suitably functionalized dyes.
In previous work, low coupling efficiencies (~ 40%) were observed for amine to
NHS-ester reactions and therefore, in Chapter 3, we investigated the use
of an improved approach relying on a bioorthogonal and highly efficient
copper catalyzed azide-alkyne cycloaddition reaction. We first enzymatically
modified the DNA substrate with a terminal alkyne using a SAM analog,
which was then clicked to an azide containing dye. This reaction showed to
be much more efficient (~ 70%) than the NHS coupling technique, although
some DNA damage was observed due to the use of a transition metal catalyst. This was partially circumvented for small, plasmid DNA with the aid
of coordinating ligands but remained a problem for larger DNA fragments.
Therefore, there is still a need for more biological compatible coupling
reactions.
In Chapter 4, distinct fluorophore coupling reactions are compared, with
the aim of increasing labeling efficiency, while maintaining DNA integrity.
Here, the amine to NHS activated ester condensation, as well as the coppercatalyzed
click reactions and a strain-promoted copper-free 1,3-dipolar
cycloaddition were compared. The copper free click reaction did not only
show to completely preserve the DNA structure, but also to be the most
efficient coupling reaction of all three.
Chapter 5 demonstrates the chemoenzymatic synthesis of a SAM analog,
containing an emissive, isomorphic nucleobase, which is also active in
DNA methylation reactions. The novel cofactor could also be generated
in situ and directly be used for methylating plasmid DNA, which can
overcome cofactor stability issues. Spectroscopic analysis showed that
the SAM analog and its demethylated product were fluorescently distinct,
thus potentially providing a tool to monitor methyl transfer reactions using
optical techniques.
During this graduate study, I have synthesized several new SAM analogs,
which contain functional groups suitable for enzymatic DNA tagging and
further fluorescent labeling. Both the efficiency of the process and its impact
on the structural integrity of the DNA were investigated, as both factors are
key to obtaining a useful and reliable DNA map. Future work in this area will
need to concentrate on identifying effective synthetic pathways for SAM
analogs containing functionalizable groups or built in fluorophores.
To conclude, fluorescence-based techniques have become essential in
modern science. Regardless of the amazing advances in instrumentation
and its sophistication, these tools ultimately rely on the availability of
bright fluorophores and on practical methods for incorporating them into
biomolecules. While numerous approaches have been advanced, harvesting
the efficiency, specificity and mild operating conditions of enzymes for
creating unnaturally-modified biomolecules has emerged as an appealing
modern direction. Indeed, recent years have seen incredible progress in
bringing together the capacity of enzymes with synthetic chemists’ creativity
and skillful art of preparing novel cofactors. This direction is not without
challenges, but when successful it can alter the course of science. Superresolution
optical DNA mapping appears to be one such direction, which is
on the verge of becoming a game-changing tool for genetic analysis.
Description: /
Table of Contents: Chapter 1: Introduction
1.1 Abstract ....................................................................................................1
1.2 Introduction..............................................................................................2
1.3 Labeling strategies using SAM dependent MTases..............................2
1.3.1 SAM analogs.............................................................................................4
1.3.2 Stability of SAM analogs..........................................................................8
1.3.3 Synthesis of SAM analogs.....................................................................12
1.3.4 Methyltransferases.................................................................................15
1.4 Current and future applications ...........................................................21
1.4.1 Selective enrichment of biomolecules.................................................21
1.4.2 Genomic analysis ...................................................................................23
1.4.3 Epigenetic analysis.................................................................................28
1.5 Conclusions and future outlook............................................................33
1.6 References..............................................................................................34
Chapter 2: Chemical synthesis of SAM analogs and their use in
DNA labeling experiments
2.1 Abstract ..................................................................................................45
2.2 Introduction ..........................................................................................45
2.3 Synthesis of SAM analogs ....................................................................46
2.4 Restriction assay as a screening for DNA labeling..............................61
2.4.1 Introduction to transalkylation reaction on DNA.................................61
2.4.2 Results restriction assays of SAM analogs sythesized via route A.....62
2.4.2 Results restriction assays of SAM analogs sythesized via route C.....71
2.5 Conclusions............................................................................................73
2.6 References..............................................................................................74
Chapter 3: Copper catalyzed click chemistry as a tool for fluorescent DNA ..
labeling
3.1 Abstract...................................................................................................77
3.2 Introduction............................................................................................77
3.2.1 Copper catalyzed click chemistry as a choice for mTAG DNA ..............
labeling...................................................................................................80
3.2.2 AdoEnYn as a choice for CuAAC reaction...........................................82
3.3 Results.....................................................................................................82
3.3.1 Screening for DNA methyltransferase activity with the AdoEnYn ........
cofactor...................................................................................................82
3.3.2 DNA damage during the CuAAC reaction..........................................83
3.3.3 DNA deposition via molecular combing..............................................84
3.3.4 Single-molecule genomic DNA mapping............................................85
3.4 Discussion...............................................................................................87
3.4.1 A subset of methyltransferases perform DNA transalkylation with .........
AdoEnYn.................................................................................................87
3.4.2 DNA damage under optimal conditions for the CuAAC reaction.....89
3.4.3 Single-molecule genomic mapping.....................................................91
3.5 Conclusion..............................................................................................92
3.6 References..............................................................................................93
Chapter 4: Methyltransferase-directed covalent coupling of fluorophores to
DNA
4.1 Abstract...................................................................................................99
4.2 Introduction..........................................................................................100
4.3 Results and discussion.........................................................................102
4.3.1 DNA damage........................................................................................104
4.4 Conclusions..........................................................................................106
4.5 References............................................................................................107
Chapter 5: Chemoenzymatic synthesis and utilization of a SAM analog with
an isomorphic nucleobase
5.1 Abstract.................................................................................................111
5.2 Introduction..........................................................................................112
5.3 Results and discussion.........................................................................115
5.4 Fluorescence comparison of SthAM, SthAH and its precursors.........119
5.5 Conclusions..........................................................................................120
5.6 References............................................................................................121
Chapter 6: Conclusions and future perspectives
6.1 Conclusions..........................................................................................125
6.2 Future perspectives..............................................................................126
Appendix A: Chapter 1
A.1 Supporting figures............................................................................... A-1
Appendix B: Chapter 2
B.1 Materials and methods........................................................................ B-1
B.2. Synthesis of compounds...................................................................... B-1
B.3. Restriction assays............................................................................... B-23
B.3.1. General restriction assay.................................................................... B-23
B.3.2. Restriction assay after in situ formation of SAM.............................. B-23
A.3. References.......................................................................................... B-24
Appendix C: Chapter 3
B.1. Materials and methods........................................................................C-1
B.2. Supplementary Figures........................................................................C-3
B.3. References..........................................................................................C-11
Appendix D: Chapter 4
D.1. Materials and methods........................................................................D-1
D.2. Experimental procedures....................................................................D-1
D.2.1. Synthesis of ADIBO Rhodamine B......................................................D-1
D.2.2. Preparation of sequence-specifically modified DNA........................D-2
D.2.3. Fluorescent labeling using the amine-to-NHS-ester coupling.........D-2
D.2.4. Fluorescent labeling using the CuAAC reaction...............................D-2
D.2.5. Fluorescent labeling using the SPAAC reaction and the solvent effect D-3
D.2.6. Fluorescence microscopy setup..........................................................D-3
D.2.7. Sample preparation.............................................................................D-3
D.2.8. Imaging.................................................................................................D-3
D.2.9. Data analysis.........................................................................................D-4
D.2.10. AFM measurements.............................................................................D-4
D.3. Supplementary Figures........................................................................D-5
D.4. References............................................................................................D-8
Appendix D: Chapter 5
E.1. Materials and methods.......................................................................................... E-1
E.2. Synthetic procedures............................................................................................. E-1
E.2.1. General.................................................................................................. E-1
E.2.2. Synthesis of ClDthA............................................................................... E-1
E.2.3. Synthesis of SthAH................................................................................ E-2
E.3. Enzymatic protocols............................................................................................... E-2
E.3.1. Expression and purification of SalL.................................................... E-2
E.3.2. Chemoenzymatic synthesis and purification of SthAM...................... E-3
E.4. Enzymatic methylation-restriction experimentsE-............................................... E-4
E.4.1. General.................................................................................................. E-4
E.4.2. Time-course methylation-restriction assay........................................ E-4
E.4.3. One-pot synthesis/methylation/restriction........................................ E-4
E.5. Fluorescence experiments.................................................................................... E-5
E.6. Supplementary figures........................................................................................... E-6
E.7. References............................................................................................................ E-10
Chemical safety................................................................................................................ I
List of publications, presentations and posters...................................................... III
URI: 
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Molecular Design and Synthesis
Molecular Imaging and Photonics

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