Synthesis characterization and application of novel 5'-capped RNAs:
Gespeichert in:
| 1. Verfasser: | |
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
Heidelberg
2019
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| Schlagworte: | |
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| Beschreibung: | X, 150 Seiten Illustrationen 30 cm |
Internformat
MARC
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| 100 | 1 | |a Abele, Florian |e Verfasser |0 (DE-588)1112212280 |4 aut | |
| 245 | 1 | 0 | |a Synthesis characterization and application of novel 5'-capped RNAs |c presented by Diplomchemiker Florian Abele |
| 264 | 1 | |a Heidelberg |c 2019 | |
| 300 | |a X, 150 Seiten |b Illustrationen |c 30 cm | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |b Dissertation |c Ruprecht-Karls-Universität Heidelberg |d 2019 | ||
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
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Datensatz im Suchindex
| _version_ | 1804180608847446016 |
|---|---|
| adam_text | CONTENTS
CONTENTS
...................................................................................................................................................
I
SCIENTIFIC
CONTRIBUTIONS
AND
PUBLICATIONS
...............................................................................................
V
ACKNOWLEDGMENTS
................................................................................................................................
VII
SUMMARY
.............................................................................................................................................
VIII
ZUSAMMENFASSUNG
..................................................................................................................................
IX
1
INTRODUCTION
.....................................................................................................................................
1
1.1
INVESTIGATION
OF
NUCLEIC
ACIDS
..................................................................................................
1
1.2
NUCLEIC
ACID
STRUCTURE
COMPOSITION
AND
BIO
SYNTHESIS
............................................................
2
1.3
NUCLEOTIDES
AND
NUCLEOTIDE
CONJUGATES
IN
NATURE
........................................................
3
1.4
IMPORTANT
PROPERTIES
OF
PHOSPHATE
(ESTERS)
FOR
BIOLOGICAL
PROCESSES
....................................
5
1.5
CAP
STRUCTURES
IN
PROKARYOTES
AND
EUKARYOTES
.......................................................................
5
1.6
APPROACHES
FOR
THE
BIOCATALYTIC
SYNTHESIS
OF
5 COFACTOR
MODIFIED
RNAS
...............................
7
1.6.1
IN
VITRO
TRANSCRIPTION
(IVI)
WITH
NON-CANONICAL
INITIATING
NUCLEOTIDES
(NCINS)...............
7
1.6.2
RIBOZYME
CATALYZED
RNA
5 -END
MODIFICATION
................................................................
8
1.7
DECAPPING
ENZYMES.................................................................................................................
8
1.8
METHODS
FOR
THE
ANALYSIS
OF
RNA
S -PROCESSING
....................................................................
10
1.9
FLUORESCENT
NUCLEOBASES
FOR
THE
STUDY
OF
NUCLEIC
ACIDS........................................................
10
1.10
RELEVANT
NUCLEOTIDE
AND
OLIGONUCLEOTIDE
CHEMISTRY
.............................................................
12
1.10.1
SYNTHESIS
OF
NUCLEOSIDE
POLYPHOSPHATES
AND
ENZYME
COFACTORS
...................................
12
1.10.2
POLYNUCLEOTIDE
SYNTHESIS
...............................................................................................
15
1.10.3
SOLID-PHASE
OLIGONUCLEOTIDE
SYNTHESIS
(SPOS)
................................................................
17
1.10.4
STRUCTURAL
AND
CHEMICAL
PROPERTIES
OF
DESCRIBED
PHOSPHORUS
COMPOUNDS
..................
21
2
OBJECTIVE
.......................................................................................................................................
23
3
RESULTS
AND
DISCUSSION..............................................................................................
25
3.1
CHEMICAL
SYNTHESIS
OF
5 -NAD-RNA
.......................................................................................
25
3.2
A
FLUORESCENCE
BASED
TECHNIQUE
TO
MONITOR
RNA
5 -END
PROCESSING
...................................
30
3.2.1
SYNTHESIS
OF
FLUORESCENT
8-SUBSTITUTED-NAD
DERIVATIVES
...............................................
31
3.2.2
SYNTHESIS
OF
8-(FURAN-2-YL)-NAD-RNA
(
FUR
NAD-RNA)
......................................................
33
3.2.3
FLUORESCENCE
PROPERTIES
OF
FUR
AMP-RNAS
AND
FUR
NAD-RNAS
.........................................
39
3.2.4
MONITORING
ENZYMATIC
ACTIVITY
AND
ENZYME
KINETICS
.....................................................
41
3.2.5
DETERMINATION
OF
KINETIC
PARAMETERS
............................................................................
42
3.2.6
SCREENING
AND
IDENTIFICATION
OF
DECAPPING
ENZYMES.....................................................
44
3.2.7
SEQUENCE
DEPENDENCY
OF
ENZYMATIC
5 -NAD-RNA
PROCESSING......................................
45
3.3
SYNTHESIS
OF
M
7
G-CAPPED
RNAS
.............................................................................................
47
3.4
SYNTHESIS
OF
SUBSTRATES
FOR
THE
CO-CRYSTALLISATION
WITH
NUDC
AND
RNASEE
............................
53
3.4.1
SYNTHESIS
OF
A
NON-HYDROLYZABLE
NAD
ANALOG................................................................
53
3.4.2
SYNTHESIS
OF
SHORT
NAD-RNAS
........................................................................................
56
3.5
APPROACHES
FOR
A
CAPTURE
PROTOCOL
OF
5 -UDP-GLCNAC
MODIFIED
RNAS
.................................
57
3.5.1
SYNTHESIS
OF
S -GICNAC-PP-RNAS
...................................................................................
57
3.5.2
CHEMO-ENZYMATIC
CAPTURE
APPROACHES
OF
PROTEINS
AND
RNAS
......................................
61
3.5.3
ENZYMATIC
LINKAGE
OF
AN
AZIDO
SUGAR
(GALNAZ)
TO
UDP-GICNAC
AND
ADP-GICNAC
..........
62
3.5.4
ENZYMATIC
MODIFICATION
OF
5 -GLCNAC-RNAS
UTILIZING
MURA
AND
MURB
...........................
63
3.5.5
A
STRATEGY
FOR
CHEMICAL
OXIDATION
OF
UDP-GICNAC
RNAS
AND
ITS
CONJUGATION
................
68
3.5.6
UTILIZATION
OF
GLMU
FOR
PUTATIVE
S -GICNAC-RNA
SYNTHESIS
...........................................
71
3.5.7
EVALUATION
OF
ENZYMATIC
PYROPHOSPHATASE
ACTIVITY
UPON
UDP-GICNAC
AND
GICNAC-PP-RNA
..............................................................................................................................
72
3.6
FURTHER
APPROACHES
FOR
THE
SYNTHESIS
OF
RNA
5 -CAP
STRUCTURES
...........................................
73
4
CONCLUSION
AND
OUTLOOK
................................................................................................................
80
4.1
SYNTHESIS
OF
NAD-RNA
...........................................................................................................
80
4.2
FLUORESCENCE
BASED
MONITORING
OF
5 -NAD-RNA
AND
5 -M
7
G-RNA
DECAPPING
......................
80
4.2.1
ACTIVITY
OF
CD38
UPON
5 -NAD-RNA...............................................................................
81
4.3
CHEMO-ENZYMATIC
CAPTURE
OF
UDP-GICNAC
RNAS
..................................................................
81
4.3.1
IN
VITRO
TRANSCRIPTION
WITH
UDP-GICNAC/ADP-GICNAC
....................................................
81
4.3.2
UTILIZATION
OF
GALTL
........................................................................................................
82
4.3.3
UTILIZATION
OF
MURA
AND
MURB
.......................................................................................
82
4.3.4 PERIODATE
TREATMENT
OF
SYNTHESIZED
GICNAC-RNAS
(NAD-RNAS)
....................................
83
4.4 APPROCHES
FOR
THE
CHEMICAL
SYNTHESIS
OF
DIFFERENT
NUCLEOTIDE
5 -RNA
MODIFICATIONS
..........
84
4.4.1
GENERAL
5 -P-RNA
ACTIVATION
AND
COUPLING
....................................................................
84
4.4.2
CHEMICAL
SYNTHESIS
OF
FAD-RNA
....................................................................................
84
4.4.3
SYNTHESIS
OF
5 -DAG-RNA
...............................................................................................
85
5
EXPERIMENTAL
PART
.........................................................................................................................
86
5.1
HPLC
METHODS
...........................................................................................................................
86
II
5.2
PREPARATION
OF
MURA,
MURB
AND
GLMU
.................................................................................
87
5.2.1
PCR
AMPLIFICATION
..........................................................................................................
87
5.2.2
PREPARATION
FOR
LIGATION
.................................................................................................
87
5.2.3
LIGATION
..........................................................................................................................
87
5.2.4
CLONING
AND
CONFIRMATION
OF
CORRECT
PLASMIDS
..............................................................
87
5.2.5
PROTEIN
EXPRESSION.........................................................................................................
88
5.2.6
PROTEIN
PURIFICATION
.......................................................................................................
88
5.3
OLIGONUCLEOTIDE
SYNTHESIS
.....................................................................................................
89
5.3.1
SOLID-PHASE
SYNTHESIS
OF
(MODIFIED)
OLIGONUCLEOTIDES
AND
DINUCLEOTIDES
.....................
89
5.4
EXONUCLEASE
DIGEST
(PL)
AND
LC-MS
ANALYSIS
OF
S -NAD-RNA
AND
S -PPPRNA
.......................
92
5.5
PREPARATION
OF
5 -MONOPHOSPHORYLATED
RNA
BY
POLYPHOSPHATASE
REACTION
........................
93
5.6
IN-VITRO
TRANSCRIPTION
(IVI)
WITH
NON-CANONICAL
INITIATOR
NUCLEOTIDES
....................................
93
5.6.1
DNA-TEMPLATES
FOR
IN
VITRO
TRANSCRIPTION
OF
25MERS:
....................................................
93
5.6.2
SEQUENCE
OF
RNA1
(107MER)
.........................................................................................
93
5.6.3
SYNTHESIZED
(IVT)
5 -(N-ACETYL-GLUCOSAMINE
DIPHOSPHATE)
MODIFIED
RNAS
(S -GICNAC-PP-RNAS)
.....................................................................................................................
94
5.7
ENZYMATIC
REACTIONS
WITH
SYNTHESIZED
OR
PURCHASED
SUBSTRATES
..........................................
94
5.7.1
MEASUREMENT
OF
FLUORESCENCE
TIME-COURSE
AND
FLUORESCENCE
SPECTRA
........................
94
5.7.2
MURA
REACTIONS
[162]
....................................................................................................
95
5.7.3
ADP-GICNAC-EP
SYNTHESIS
..............................................................................................
95
5.7.4
SYMTHESIS
OF
EP-GICNAC-PP-A-RNAS
..............................................................................
96
5.7.5
MURA
AND
MURB
ONE
POT
REACTIONS
[162]
......................................................................
96
5.7.6
GLMU
..............................................................................................................................
97
5.8
CHEMICAL
SYNTHESIS
................................................................................................................
97
5.8.1
IO4-OXIDATION
OF
NUCLEOTIDES
AND
RNAS
.........................................................................
97
5.8.2
GENERAL
REACTION
PRESCRIPTIONS
......................................................................................
97
5.8.3
TRIETHYLAMMONIUM
BICARBONATE
BUFFER
(3
M,
PH
8)
......................................................
98
5.8.4
NMN
ACTIVATION
FOR
5 -NAD-RNA...................................................................................
98
5.8.5
SYNTHESIS
OF
8-AR-SUBSTITUTED
AMP
AND
NAD-DERIVATIVES
...........................................
100
5.8.6
SYNTHESIS
OF
8-(FURAN-2-YL)-ADENOSINE
3 -PHOSPHORAMIDITE
........................................
102
5.8.7
SYNTHESIS
AND
ACTIVATION
OF
7-METHYL-GUANOSINE-DIPHOSPHATE
(M
7
GPP)
[124]
...........
106
5.8.8
ATTEMPTED
SYNTHESIS
OF
A
FLUORESCENT
M
7
G
CAP...........................................................
108
5.8.9
SYNTHESIS
OF
NON-HYDROLYZABLE
NAD
(PCP-NAD,
22)
....................................................
110
III
5.8.10
SYNTHESIS
OF
ADP-GICNAC
.............................................................................................
114
5.8.11
SYNTHESIS
OF
FURTHER
5 -RNA
MODIFICATIONS
..................................................................
118
OAT-NMN
.....................................................................................................................................
118
6
APPENDIX
.......................................................................................................................................
124
6.1
LIST
OF
ABBREVIATIONS
............................................................................................................
124
6.2
LIST
OF
TECHNICAL
EQUIPMENT
.................................................................................................
126
6.3
ADDITIONAL
MASS
SPECTRA
......................................................................................................
127
6.3.1
ESI-TOF
MASS
SPECTRA
OF
FLUORESCENT
8-SUBSTITUTED
NUCLEOTIDES
.................................
127
6.3.2
HRMS
(ESI)
AND
HPLC
OF
SYNTHESIZED
DINUCLEOTIDES
....................................................
128
6.3.3
CHARACTERISATION
OF
CD38
REACTION
PRODUCTS
-
S -ADPR-RNA
......................................
129
6.4
FLUORESCENCE
SPECTRA
OF
FUR
A-RNAS
AND
FUR
NAD-RNAS
...........................................................
130
6.5
HPLC
CHROMATOGRAMS
..........................................................................................................
131
6.5.1
HPLC
CHROMATOGRAMS
OF
INITIATOR
NUCLEOTIDES
AND
IVTS
..............................................
131
6.5.2
GICNAC-RNAS
AS
SUBSTRATES
FOR
MURA
..........................................................................
133
6.5.3
UDP-GICNAC
AND
GLCNAC-3MER
IN
PRESENCE
OF
NUDIX
HYDROLASES
...............................
133
6.6
CLONING
.................................................................................................................................
134
6.6.1
MURA
(47.4
KDA)..........................................................................................................
134
6.6.2
MURB
(41.9
KDA)
..........................................................................................................
135
6.6.3
GLMU
(50.1
KDA)..........................................................................................................
135
6.7
NMR-SPECTRA
OF
SELECTED
SYNTHESIZED
COMPOUNDS
.............................................................
137
7
REFERENCES...................................................................................................................................
142
IV
|
| any_adam_object | 1 |
| author | Abele, Florian |
| author_GND | (DE-588)1112212280 |
| author_facet | Abele, Florian |
| author_role | aut |
| author_sort | Abele, Florian |
| author_variant | f a fa |
| building | Verbundindex |
| bvnumber | BV046213712 |
| ctrlnum | (OCoLC)1125191714 (DE-599)DNB1194245749 |
| discipline | Chemie / Pharmazie |
| format | Thesis Book |
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| genre_facet | Hochschulschrift |
| id | DE-604.BV046213712 |
| illustrated | Illustrated |
| indexdate | 2024-07-10T08:38:28Z |
| institution | BVB |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-031592554 |
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| open_access_boolean | |
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| owner_facet | DE-355 DE-BY-UBR |
| physical | X, 150 Seiten Illustrationen 30 cm |
| publishDate | 2019 |
| publishDateSearch | 2019 |
| publishDateSort | 2019 |
| record_format | marc |
| spelling | Abele, Florian Verfasser (DE-588)1112212280 aut Synthesis characterization and application of novel 5'-capped RNAs presented by Diplomchemiker Florian Abele Heidelberg 2019 X, 150 Seiten Illustrationen 30 cm txt rdacontent n rdamedia nc rdacarrier Dissertation Ruprecht-Karls-Universität Heidelberg 2019 (DE-588)4113937-9 Hochschulschrift gnd-content B:DE-101 application/pdf http://d-nb.info/1194245749/04 Inhaltsverzeichnis DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=031592554&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Abele, Florian Synthesis characterization and application of novel 5'-capped RNAs |
| subject_GND | (DE-588)4113937-9 |
| title | Synthesis characterization and application of novel 5'-capped RNAs |
| title_auth | Synthesis characterization and application of novel 5'-capped RNAs |
| title_exact_search | Synthesis characterization and application of novel 5'-capped RNAs |
| title_full | Synthesis characterization and application of novel 5'-capped RNAs presented by Diplomchemiker Florian Abele |
| title_fullStr | Synthesis characterization and application of novel 5'-capped RNAs presented by Diplomchemiker Florian Abele |
| title_full_unstemmed | Synthesis characterization and application of novel 5'-capped RNAs presented by Diplomchemiker Florian Abele |
| title_short | Synthesis characterization and application of novel 5'-capped RNAs |
| title_sort | synthesis characterization and application of novel 5 capped rnas |
| topic_facet | Hochschulschrift |
| url | http://d-nb.info/1194245749/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=031592554&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT abeleflorian synthesischaracterizationandapplicationofnovel5cappedrnas |
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