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Click chemistry for the modification of oligonucleotides and their applications
Click chemistry for the modification of oligonucleotides and their applications
This PhD thesis reports the published work done in the laboratories of baselick GmbH and the Ludwig Maximilians Universität (LMU). baseclick GmbH was founded by Prof. Dr. Thomas Carell (LMU) in collaboration with the chemical company BASF in 2008. Main focus of baseclick is the usage of click chemistry for modification of nucleic acids. In the described research work click chemistry is applied to oligonucleotides and in particular to DNA nanostructures, to mRNA to be then used in drug development and to produce highly labeled probes for fluorescent in situ hybridization. Continuing the previous work done at baseclick, at first click chemistry was applied on DNA nanostructures. In this field DNA, is not used as a carrier of genetic information but as material for the production of structures with different size, geometry and shape. The main concept behind the technology is based on Watson and Crick base pairing interactions, which bring portions of a ssDNA to hybridize with a complementary sequence, usually of another ssDNA strand, to form a rigid dsDNA helix. This is used in the rational design of the sequences to form complex structures with nanometric precision. DNA nanostructures and click chemistry were used to find an alternative to the current state of the art methods for gene synthesis in vitro. To date enzymatic synthesis of long DNA fragment is the method of choice since solid phase synthesis is not suitable for very long sequences. Enzymatic synthesis approaches are based on the activity of either DNA polymerases or DNA ligases reactions, but those methods suffer from some limitations: e.g. in the case of ligases the final gene is assembled by overlapping of strands then ligated to form a longer fragment, but it starts to be insufficient when a big number of strands need to be ligated together. With DNA polymerases the final product is formed by different cycles of the enzyme in a multiple step assembly, with the limitation due to the mispriming and formation of secondary structures which then lead to errors. Therefore in here, in collaboration with the group of Prof. Dr. Tom Brown from University of Oxford, a method was developed where, with the help of the DNA origami technique, ligase activity is replaced by chemical ligation, in this case click chemistry. 14 oligonucleotides were designed and synthesized with a 5’-terminal azide and 3’-terminal alkyne. The oligos where then preorganized in a DNA nanostructure to bring the alkyne and azide in close proximity and, most importantly, in a selective order. In this way after the click reaction occurs, the full length of the defined sequence is established, with a bio-compatible triazole linkage replacing the phosphate bond at the point of the oligo connections. In a second project click chemistry was used to stabilize a DNA nanostructure, in this case composed of 24 different interlocked oligonucleotides, and at the same time achieve selective labeling with biotin molecules in a one pot reaction. This work was done in collaboration with the group of Prof. Dr. Silvia Biocca of University of Rome Tor Vergata. DNA nanostructures thanks to their properties such as bio-compatibility, non-toxicity and bio-degradability have been used for different applications: e.g. drug delivery, nanocontainer, cellular biosensor and in vivo imaging. Anyhow, crucial for these applications is the understanding of how different DNA nanostructure enter the mammalian cells. For this reason, in this work five different topological configurations and functionalizations, with size varying from 8 to 80 nm and shape from tetrahedral, octahedral, cylindrical, square box and rectangular, have been investigated for their ability to interact with the scavenger receptor LOX-1, which overexpression has been associated with tumor development in many cancer cells. Inspired by the big success that mRNA therapy has in the last decade, methods to enable modification of very long RNA oligonucleotides, such as mRNA, were established using click chemistry. In vitro transcribed (IVT) mRNA, consisting in mRNA produced by RNA polymerases from a DNA template, is nowadays considered to be a valid candidate for a novel class of drugs. It was already demonstrated to be efficient in several diseases including: vaccination, protein replacement and cancer therapy. Indeed nowadays mRNA is playing a central role in vaccination programs against SARS-Cov-2 pandemic. The main idea behind the mRNA therapy is to provide IVT mRNA to the patients to help them developing their own cure. For example in vaccination, mRNA coding for a specific viral antigen is used to produce an immune response leading to the immunogenicity. Besides stability issues deriving from using RNA molecule as drugs, another problem arises from the cellular delivery of such molecules. Indeed, delivery to a specific target is still an unsolved problem. To date the mRNA is delivered to patients using lipid nanoparticle (LNPs) to act as carrier and at the same time as a barrier from the extracellular environment. Recent studies demonstrate however that the usage of LNP is not the ideal method to deliver mRNA. It has been proven to be less efficient in vivo than what was observed in vitro. Especially in living organisms the main destination is the liver, which is often not the final target for the therapy. Also driven by the recent FDA approval of the first siRNA drug (GIVLAARITM), where the siRNA has been chemically modified using N-acetylgalactosamine molecules (GalNac) to enable efficient targeted delivery, it is described here a chemoenzymatic approach based on the incorporation of modified nucleotides bearing an alkyne or azide moiety (such as 5-ethynyl-UTP or 3’-azido-dd-ATP), that can then be labelled post-transcriptionally, using click chemistry. This method allows for example the incorporation of specific modifications inside the mRNA that would not be accepted by the RNA polymerases, e.g. targeting-molecules for specific delivery, or fluorescent dyes for tracking, thus potentially improving the biochemical properties of the mRNA. Click chemistry was also used to improve the current methods for the preparation of probes used in fluorescent in situ hybridization (FISH). FISH is a cytogenetic analysis that allows the detection and the spatial localization of specific nucleotide sequences in tissues or cells. The fluorescent probes, consisting of ssDNA, were designed to hybridize only to those parts of the target DNA/RNA with a high degree of complementarity. Then by utilizing fluorescent microscopy it was possible to localize where the fluorescent probes are hybridized. This technique is largely used for diagnosis of genetic abnormalities such as gene fusion, aneuploidy, loss of chromosomal regions, detection of oncogenes and diagnosis of viral infections and to date it can also detect other targets such RNA (mRNA) in cells and tissue samples. The probes can be prepared in various ways, such as nick translation, random priming, PCR, end labelling or NHS-ester chemistry. Most of the probes, especially for RNA detection, are composed of ssDNA which are approximately 20-25 nt long, conjugated to a fluorophore via coupling of an amino group introduced at the 3’ end and an activated ester of the fluorophore. Finally a method for preparation of mRNA FISH probes based on click chemistry is described, where each probe contains three fluorophore instead of a single one, thus giving an increment of fluorescent yields and allowing microscopy analysis without the need of special deconvolution software. Furthermore this allows the detection using fluorescent activated cell sorting (FACS) devices. Enabling FACS analysis is of outmost importance especially for clinical studies, where up to now the detection of specific mRNA or chromosomes sequences is still done manually by clinicians, analyzing all the samples through visual inspection.
Click chemistry, oligonucleotides, mRNA modification, Gene synthesis, FISH
Croce, Stefano
2021
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Croce, Stefano (2021): Click chemistry for the modification of oligonucleotides and their applications. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

This PhD thesis reports the published work done in the laboratories of baselick GmbH and the Ludwig Maximilians Universität (LMU). baseclick GmbH was founded by Prof. Dr. Thomas Carell (LMU) in collaboration with the chemical company BASF in 2008. Main focus of baseclick is the usage of click chemistry for modification of nucleic acids. In the described research work click chemistry is applied to oligonucleotides and in particular to DNA nanostructures, to mRNA to be then used in drug development and to produce highly labeled probes for fluorescent in situ hybridization. Continuing the previous work done at baseclick, at first click chemistry was applied on DNA nanostructures. In this field DNA, is not used as a carrier of genetic information but as material for the production of structures with different size, geometry and shape. The main concept behind the technology is based on Watson and Crick base pairing interactions, which bring portions of a ssDNA to hybridize with a complementary sequence, usually of another ssDNA strand, to form a rigid dsDNA helix. This is used in the rational design of the sequences to form complex structures with nanometric precision. DNA nanostructures and click chemistry were used to find an alternative to the current state of the art methods for gene synthesis in vitro. To date enzymatic synthesis of long DNA fragment is the method of choice since solid phase synthesis is not suitable for very long sequences. Enzymatic synthesis approaches are based on the activity of either DNA polymerases or DNA ligases reactions, but those methods suffer from some limitations: e.g. in the case of ligases the final gene is assembled by overlapping of strands then ligated to form a longer fragment, but it starts to be insufficient when a big number of strands need to be ligated together. With DNA polymerases the final product is formed by different cycles of the enzyme in a multiple step assembly, with the limitation due to the mispriming and formation of secondary structures which then lead to errors. Therefore in here, in collaboration with the group of Prof. Dr. Tom Brown from University of Oxford, a method was developed where, with the help of the DNA origami technique, ligase activity is replaced by chemical ligation, in this case click chemistry. 14 oligonucleotides were designed and synthesized with a 5’-terminal azide and 3’-terminal alkyne. The oligos where then preorganized in a DNA nanostructure to bring the alkyne and azide in close proximity and, most importantly, in a selective order. In this way after the click reaction occurs, the full length of the defined sequence is established, with a bio-compatible triazole linkage replacing the phosphate bond at the point of the oligo connections. In a second project click chemistry was used to stabilize a DNA nanostructure, in this case composed of 24 different interlocked oligonucleotides, and at the same time achieve selective labeling with biotin molecules in a one pot reaction. This work was done in collaboration with the group of Prof. Dr. Silvia Biocca of University of Rome Tor Vergata. DNA nanostructures thanks to their properties such as bio-compatibility, non-toxicity and bio-degradability have been used for different applications: e.g. drug delivery, nanocontainer, cellular biosensor and in vivo imaging. Anyhow, crucial for these applications is the understanding of how different DNA nanostructure enter the mammalian cells. For this reason, in this work five different topological configurations and functionalizations, with size varying from 8 to 80 nm and shape from tetrahedral, octahedral, cylindrical, square box and rectangular, have been investigated for their ability to interact with the scavenger receptor LOX-1, which overexpression has been associated with tumor development in many cancer cells. Inspired by the big success that mRNA therapy has in the last decade, methods to enable modification of very long RNA oligonucleotides, such as mRNA, were established using click chemistry. In vitro transcribed (IVT) mRNA, consisting in mRNA produced by RNA polymerases from a DNA template, is nowadays considered to be a valid candidate for a novel class of drugs. It was already demonstrated to be efficient in several diseases including: vaccination, protein replacement and cancer therapy. Indeed nowadays mRNA is playing a central role in vaccination programs against SARS-Cov-2 pandemic. The main idea behind the mRNA therapy is to provide IVT mRNA to the patients to help them developing their own cure. For example in vaccination, mRNA coding for a specific viral antigen is used to produce an immune response leading to the immunogenicity. Besides stability issues deriving from using RNA molecule as drugs, another problem arises from the cellular delivery of such molecules. Indeed, delivery to a specific target is still an unsolved problem. To date the mRNA is delivered to patients using lipid nanoparticle (LNPs) to act as carrier and at the same time as a barrier from the extracellular environment. Recent studies demonstrate however that the usage of LNP is not the ideal method to deliver mRNA. It has been proven to be less efficient in vivo than what was observed in vitro. Especially in living organisms the main destination is the liver, which is often not the final target for the therapy. Also driven by the recent FDA approval of the first siRNA drug (GIVLAARITM), where the siRNA has been chemically modified using N-acetylgalactosamine molecules (GalNac) to enable efficient targeted delivery, it is described here a chemoenzymatic approach based on the incorporation of modified nucleotides bearing an alkyne or azide moiety (such as 5-ethynyl-UTP or 3’-azido-dd-ATP), that can then be labelled post-transcriptionally, using click chemistry. This method allows for example the incorporation of specific modifications inside the mRNA that would not be accepted by the RNA polymerases, e.g. targeting-molecules for specific delivery, or fluorescent dyes for tracking, thus potentially improving the biochemical properties of the mRNA. Click chemistry was also used to improve the current methods for the preparation of probes used in fluorescent in situ hybridization (FISH). FISH is a cytogenetic analysis that allows the detection and the spatial localization of specific nucleotide sequences in tissues or cells. The fluorescent probes, consisting of ssDNA, were designed to hybridize only to those parts of the target DNA/RNA with a high degree of complementarity. Then by utilizing fluorescent microscopy it was possible to localize where the fluorescent probes are hybridized. This technique is largely used for diagnosis of genetic abnormalities such as gene fusion, aneuploidy, loss of chromosomal regions, detection of oncogenes and diagnosis of viral infections and to date it can also detect other targets such RNA (mRNA) in cells and tissue samples. The probes can be prepared in various ways, such as nick translation, random priming, PCR, end labelling or NHS-ester chemistry. Most of the probes, especially for RNA detection, are composed of ssDNA which are approximately 20-25 nt long, conjugated to a fluorophore via coupling of an amino group introduced at the 3’ end and an activated ester of the fluorophore. Finally a method for preparation of mRNA FISH probes based on click chemistry is described, where each probe contains three fluorophore instead of a single one, thus giving an increment of fluorescent yields and allowing microscopy analysis without the need of special deconvolution software. Furthermore this allows the detection using fluorescent activated cell sorting (FACS) devices. Enabling FACS analysis is of outmost importance especially for clinical studies, where up to now the detection of specific mRNA or chromosomes sequences is still done manually by clinicians, analyzing all the samples through visual inspection.