Biophysical Studies of Oligonucleotides Containing Duplex Stabilising Modifications
<p>Chemically modified DNA and RNA probes have been used in numerous diagnostic (e.g. fluorogenic PCR and <em>in situ</em> hybridisation) and therapeutic (e.g. exon-skipping and antisense) applications to increase duplex stability and facilitate mismatch discrimination. There is a...
| Main Author: | |
|---|---|
| Other Authors: | |
| Format: | Thesis |
| Language: | English |
| Published: |
2016
|
| Subjects: |
| _version_ | 1797054432856244224 |
|---|---|
| author | Marafini, P |
| author2 | Brown, T |
| author_facet | Brown, T Marafini, P |
| author_sort | Marafini, P |
| collection | OXFORD |
| description | <p>Chemically modified DNA and RNA probes have been used in numerous diagnostic (e.g. fluorogenic PCR and <em>in situ</em> hybridisation) and therapeutic (e.g. exon-skipping and antisense) applications to increase duplex stability and facilitate mismatch discrimination. There is a constant need for well-studied modifications, and to this end X-pyrene was evaluated as a new cytosine analogue that combines enhanced π-stacking, hydrogen bonding and electrostatic interactions to greatly increase the stability of bulged DNA duplexes and DNA/RNA hybrid duplexes. X-pyrene is highly selective for guanine, and even more importantly duplex stability is reduced dramatically when X-pyrene or a neighbouring base is mismatched. NMR and fluorescence studies indicate that the pyrene moiety stacks within the DNA double helix. To enable easier access to this modification, a reductive amination labelling strategy was developed to produce pyrene-functionalised nucleobase analogues which allowed an easier synthesis of X-pyrene. Amino C6 dT was successfully functionalised with this methodology as well, obtaining the duplex stabilising mono- and di-labelled products. Additionally, a much simpler pyrene-modified thymidine monomer (U-pyrene) was selected for further studies. Single and multiple additions of U-pyrene were found to significantly increase the melting temperature of dsDNA without perturbing the B-DNA duplex, as shown by UV and CD spectroscopy. Interestingly, thermodynamic studies showed that four additions of U-pyrene increased the binding constant of a 14-mer oligonucleotide to its complementary strand 10000-fold when compared to the unmodified control. Two oligonucleotides containing a single addition of U-pyrene were also crystallised and their structures were studied by X-ray diffraction. To the author's knowledge these represent the first examples of X-ray crystal structures of a chemically modified pyrene oligonucleotide. They also proved to be of great interest as they highlight the capacity of U-pyrene to facilitate the production of interesting nanostructures.</p> |
| first_indexed | 2024-03-06T18:57:13Z |
| format | Thesis |
| id | oxford-uuid:12419634-0b68-4199-aeaf-aaedb8e7e6d3 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-06T18:57:13Z |
| publishDate | 2016 |
| record_format | dspace |
| spelling | oxford-uuid:12419634-0b68-4199-aeaf-aaedb8e7e6d32022-03-26T10:06:56ZBiophysical Studies of Oligonucleotides Containing Duplex Stabilising ModificationsThesishttp://purl.org/coar/resource_type/c_db06uuid:12419634-0b68-4199-aeaf-aaedb8e7e6d3ChemistryBiochemistryChemical BiologyBiophysicsEnglishORA Deposit2016Marafini, PBrown, T<p>Chemically modified DNA and RNA probes have been used in numerous diagnostic (e.g. fluorogenic PCR and <em>in situ</em> hybridisation) and therapeutic (e.g. exon-skipping and antisense) applications to increase duplex stability and facilitate mismatch discrimination. There is a constant need for well-studied modifications, and to this end X-pyrene was evaluated as a new cytosine analogue that combines enhanced π-stacking, hydrogen bonding and electrostatic interactions to greatly increase the stability of bulged DNA duplexes and DNA/RNA hybrid duplexes. X-pyrene is highly selective for guanine, and even more importantly duplex stability is reduced dramatically when X-pyrene or a neighbouring base is mismatched. NMR and fluorescence studies indicate that the pyrene moiety stacks within the DNA double helix. To enable easier access to this modification, a reductive amination labelling strategy was developed to produce pyrene-functionalised nucleobase analogues which allowed an easier synthesis of X-pyrene. Amino C6 dT was successfully functionalised with this methodology as well, obtaining the duplex stabilising mono- and di-labelled products. Additionally, a much simpler pyrene-modified thymidine monomer (U-pyrene) was selected for further studies. Single and multiple additions of U-pyrene were found to significantly increase the melting temperature of dsDNA without perturbing the B-DNA duplex, as shown by UV and CD spectroscopy. Interestingly, thermodynamic studies showed that four additions of U-pyrene increased the binding constant of a 14-mer oligonucleotide to its complementary strand 10000-fold when compared to the unmodified control. Two oligonucleotides containing a single addition of U-pyrene were also crystallised and their structures were studied by X-ray diffraction. To the author's knowledge these represent the first examples of X-ray crystal structures of a chemically modified pyrene oligonucleotide. They also proved to be of great interest as they highlight the capacity of U-pyrene to facilitate the production of interesting nanostructures.</p> |
| spellingShingle | Chemistry Biochemistry Chemical Biology Biophysics Marafini, P Biophysical Studies of Oligonucleotides Containing Duplex Stabilising Modifications |
| title | Biophysical Studies of Oligonucleotides Containing Duplex Stabilising Modifications |
| title_full | Biophysical Studies of Oligonucleotides Containing Duplex Stabilising Modifications |
| title_fullStr | Biophysical Studies of Oligonucleotides Containing Duplex Stabilising Modifications |
| title_full_unstemmed | Biophysical Studies of Oligonucleotides Containing Duplex Stabilising Modifications |
| title_short | Biophysical Studies of Oligonucleotides Containing Duplex Stabilising Modifications |
| title_sort | biophysical studies of oligonucleotides containing duplex stabilising modifications |
| topic | Chemistry Biochemistry Chemical Biology Biophysics |
| work_keys_str_mv | AT marafinip biophysicalstudiesofoligonucleotidescontainingduplexstabilisingmodifications |