Towards smart antisense and antigene therapeutics : design and synthesis of novel oligonucleotide based bioconjugates
Author: Jezowska-Herrera, Martina
Date: 2016-06-09
Location: Lecture Hall, floor 4, Novum, Hälsovägen 7, Huddinge
Time: 10.00
Department: Inst för biovetenskaper och näringslära / Dept of Biosciences and Nutrition
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Thesis (4.199Mb)
Abstract
The use of nucleic acid technology has been of great importance in life science research. In
several clinical trials patients with genetic disease are treated with help of innovations based
on oligonucleotide therapy. The use of it is unfortunately limited in efficacy due to e.g., variable
stability of oligonucleotides in biological fluids and in particular poor delivery to the site of
action. Numerous modifications, introduced to functionalize oligonucleotides improve their
properties and help to overcome the limitation connected to their uptake.
With this thesis, I present our development of novel bioconjugates and technologies for functionalization of oligonucleotides. In chapter 2 (paper I), the development of four different synthetic m3G-Cap (nuclear localization signals) constructs is described. We provide a protocol that allows the conjugation of these for derivatives to oligonucleotide using “click” reaction. As a result, novel bioconjugates equipped with constructs containing the m3G-Cap were synthesized for the investigation of nuclear delivery.
Chapter 3 (paper II) describes the synthesis of two biotinylated linkers that can be conjugated to both peptides and oligonucleotides for labeling purposes. The multi-step synthesis of the linker is followed by conjugation to oligonucleotides that was carried out on solid support as well as to a peptide in solution.
In chapter 4 (paper III), the research presented in paper I and II is combined to allow the visualization of m3G-Cap action. The biotin linkers were also conjugated with similar Cap constructs containing modifications of the triphosphate bridge. All bioconjugates were evaluated in cell assays for uptake and splice-switching and the results show that the minimal m3G-Cap-biotin construct should contain a trinucleotide in order to act as a nuclear transport signal.
Chapter 5 gives an insight into unpublished data on the development of a linker that enable multiple functionalization of oligonucleotides (ONs) with several different biologically active entities. The synthesis of the linker, together with the first oligonucleotide conjugates prepared using the protocol is reported.
In chapter 6 (paper IV), we describe the successful preparation of four different Zorro-LNA constructs with help of “click” chemistry. Their ability for DSI (double strand invasion) was tested and they proved to be able to invade into supercoiled DNA, providing a useful screening strategy when optimizing Zorro constructs directed against new anti-gene targets.
With this thesis, I present our development of novel bioconjugates and technologies for functionalization of oligonucleotides. In chapter 2 (paper I), the development of four different synthetic m3G-Cap (nuclear localization signals) constructs is described. We provide a protocol that allows the conjugation of these for derivatives to oligonucleotide using “click” reaction. As a result, novel bioconjugates equipped with constructs containing the m3G-Cap were synthesized for the investigation of nuclear delivery.
Chapter 3 (paper II) describes the synthesis of two biotinylated linkers that can be conjugated to both peptides and oligonucleotides for labeling purposes. The multi-step synthesis of the linker is followed by conjugation to oligonucleotides that was carried out on solid support as well as to a peptide in solution.
In chapter 4 (paper III), the research presented in paper I and II is combined to allow the visualization of m3G-Cap action. The biotin linkers were also conjugated with similar Cap constructs containing modifications of the triphosphate bridge. All bioconjugates were evaluated in cell assays for uptake and splice-switching and the results show that the minimal m3G-Cap-biotin construct should contain a trinucleotide in order to act as a nuclear transport signal.
Chapter 5 gives an insight into unpublished data on the development of a linker that enable multiple functionalization of oligonucleotides (ONs) with several different biologically active entities. The synthesis of the linker, together with the first oligonucleotide conjugates prepared using the protocol is reported.
In chapter 6 (paper IV), we describe the successful preparation of four different Zorro-LNA constructs with help of “click” chemistry. Their ability for DSI (double strand invasion) was tested and they proved to be able to invade into supercoiled DNA, providing a useful screening strategy when optimizing Zorro constructs directed against new anti-gene targets.
List of papers:
I. Malgorzata Honcharenko, Joanna Romanowska, Margarita Alvira, Martina Jezowska, Mikael Kjellgren, C. I. Edvard Smith and Roger Strömberg; Capping of oligonucleotides with “clickable” m3G-CAPs. RSC Advances, 2012, 2, 12949
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II. Martina Jezowska, Joanna Romanowska, Burcu Bestas, Ulf Tedebark, Malgorzata Honcharenko; Synthesis of biotin linkers with the activated triple bond donor [p-(Npropynoylamino)toluic acid] (PATA) or efficient biotinylation of peptides and oligonucleotides. Molecules, 2012, 17, 14174
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III. Malgorzata Honcharenko, Burcu Bestas, Martina Jezowska, Błażej A. Wojtczak, Pedro Moreno, Joanna Romanowska, Susanna M. Bächle, Edward Darzynkiewicz, Jacek Jemielity, C. I. Edvard Smith, Roger Strömberg; Synthetic m3G-CAP attachment necessitates a minimum trinucleotide cinstituent to be recognised as a Nuclear Import Signal. [Manuscript]
IV. Olof I. Gissberg, Martina Jezowska, Eman M. Zaghloul, N.I. Bungsua, Roger Strömberg, C. I. Edvard Smith, Karin E. Lundin, Malgorzata Honcharenko; Fast and Efficient Synthesis of Zorro-LNA Type 3’-5'-5'-3’ Oligonucleotide Conjugates via parallel in-situ stepwise conjugation. Org. Biomol. Chem., 2016,14, 3584
Fulltext (DOI)
Pubmed
View record in Web of Science®
I. Malgorzata Honcharenko, Joanna Romanowska, Margarita Alvira, Martina Jezowska, Mikael Kjellgren, C. I. Edvard Smith and Roger Strömberg; Capping of oligonucleotides with “clickable” m3G-CAPs. RSC Advances, 2012, 2, 12949
Fulltext (DOI)
View record in Web of Science®
II. Martina Jezowska, Joanna Romanowska, Burcu Bestas, Ulf Tedebark, Malgorzata Honcharenko; Synthesis of biotin linkers with the activated triple bond donor [p-(Npropynoylamino)toluic acid] (PATA) or efficient biotinylation of peptides and oligonucleotides. Molecules, 2012, 17, 14174
Fulltext (DOI)
Pubmed
View record in Web of Science®
III. Malgorzata Honcharenko, Burcu Bestas, Martina Jezowska, Błażej A. Wojtczak, Pedro Moreno, Joanna Romanowska, Susanna M. Bächle, Edward Darzynkiewicz, Jacek Jemielity, C. I. Edvard Smith, Roger Strömberg; Synthetic m3G-CAP attachment necessitates a minimum trinucleotide cinstituent to be recognised as a Nuclear Import Signal. [Manuscript]
IV. Olof I. Gissberg, Martina Jezowska, Eman M. Zaghloul, N.I. Bungsua, Roger Strömberg, C. I. Edvard Smith, Karin E. Lundin, Malgorzata Honcharenko; Fast and Efficient Synthesis of Zorro-LNA Type 3’-5'-5'-3’ Oligonucleotide Conjugates via parallel in-situ stepwise conjugation. Org. Biomol. Chem., 2016,14, 3584
Fulltext (DOI)
Pubmed
View record in Web of Science®
Institution: Karolinska Institutet
Supervisor: Honcharenko, Malgorzata
Issue date: 2016-05-16
Rights:
Publication year: 2016
ISBN: 978-91-7676-294-3
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