The application of thiazole orange and artificial backbone modifications in the synthesis of fluorescent nucleic acids probes
<p>Thiazole orange (TO) is an asymmetric cyanine dye used in nucleic acid detection. It has wide applications due to its DNA-intercalating properties, resulting in an “on-off” fluorescent response upon binding to complementary nucleic acids. This thesis describes the use of TO in various fluor...
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| Other Authors: | |
| Format: | Thesis |
| Language: | English |
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2019
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| _version_ | 1811139155689734144 |
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| author | Klimkowski, P |
| author2 | Brown, T |
| author_facet | Brown, T Klimkowski, P |
| author_sort | Klimkowski, P |
| collection | OXFORD |
| description | <p>Thiazole orange (TO) is an asymmetric cyanine dye used in nucleic acid detection. It has wide applications due to its DNA-intercalating properties, resulting in an “on-off” fluorescent response upon binding to complementary nucleic acids. This thesis describes the use of TO in various fluorescent probe systems.</p>
<p>Incorporation of TO as a nucleobase surrogate into a modified FIT (forced intercalation) probe design via an L-threoninol acyclic backbone by amide bond formation was unsuccessful even after extensive synthetic effort. This issue was solved by introduction an azide functional group into probe sequence and efficient CuAAC labelling. The fluorogenic properties of these probes were not suitable to justify further development of the L-threoninol backbone, although this convenient synthetic route could be applied for new acyclic scaffold for FIT design.</p>
<p>New thymine nucleobase alkyne-amine modifications with various lengths of amine linker (AP-C3 and AP-C6 as phosphoramidites) were developed for the orthogonal labelling of DNA at single thymine sites using amide bond formation and CuAAC click chemistry in a single tube labelling protocol. These novel monomers were investigated in fluorescence resonance energy transfer (FRET) oligonucleotide probes with TO as the FRET donor dye, and thermal duplex melting studies were carried out showing beneficial increase of Tm with TO, where chosen reporter dye caused destabilisation . These new modifications provide an effective platform for highly efficient transfer of energy between donor and receptor fluorophore. However, undesirably high fluorescence was observed in the single stranded probe which could be improved by double incorporation of the AP-C3modification and careful choice of a reporter dye. Thermal melting studies showed modest increase of Tm for unlabelled modification and addition of TO did not results in high additional stabilizing effect. Additionally, this modification could be used as a simple building block for incorporation of a TO intercalator and an additional non-fluorescent label into oligonucleotides.</p>
<p>Investigation of the mode of attachment of thiazole orange to oligonucleotides (via its benzothiazole or quinoline moiety), and its point of attachment (nucleobase (major groove) or sugar (minor groove) were explored in several sequence contexts. Obtained results clearly shows preference of major grove (C6, PA) with TO attached via benzothiazole and minor groove (AE) with quinoline attached TO, where best fluorescence properties were achieved for pyrimidines (T and C) as a surrounding bases where purines, especially guanine gives unfavourable fluorescence enhancements. In addition, 2′-OMe sugar modification of thiazole orange probes resulted in a particularly high fluorescent enhancement and very favourable quantum yields on duplex formation. This provides a simple and effective system for detection of DNA and RNA and puts our design on par with more complex systems involving TO, including ECHO probes (exciton-controlled hybridization-sensitive fluorescent oligonucleotide probes, Okamoto); and FIT probes (forced intercalation probes, Seitz). Interestingly, the labelling of amino-modified oligonucleotides with TO-NHS ester is rapid (10 minutes at room temperature), and we were able to label oligonucleotide probes with multiple TO units in almost quantitative yields.</p> |
| first_indexed | 2024-09-25T04:01:36Z |
| format | Thesis |
| id | oxford-uuid:522beacb-6f6c-4d0b-b3cc-b3724c104412 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-09-25T04:01:36Z |
| publishDate | 2019 |
| record_format | dspace |
| spelling | oxford-uuid:522beacb-6f6c-4d0b-b3cc-b3724c1044122024-05-02T09:53:42ZThe application of thiazole orange and artificial backbone modifications in the synthesis of fluorescent nucleic acids probesThesishttp://purl.org/coar/resource_type/c_db06uuid:522beacb-6f6c-4d0b-b3cc-b3724c104412Nucleic acid probesChemical biologyChemistryRNAOligonucleotidesEnglishHyrax Deposit2019Klimkowski, PBrown, T<p>Thiazole orange (TO) is an asymmetric cyanine dye used in nucleic acid detection. It has wide applications due to its DNA-intercalating properties, resulting in an “on-off” fluorescent response upon binding to complementary nucleic acids. This thesis describes the use of TO in various fluorescent probe systems.</p> <p>Incorporation of TO as a nucleobase surrogate into a modified FIT (forced intercalation) probe design via an L-threoninol acyclic backbone by amide bond formation was unsuccessful even after extensive synthetic effort. This issue was solved by introduction an azide functional group into probe sequence and efficient CuAAC labelling. The fluorogenic properties of these probes were not suitable to justify further development of the L-threoninol backbone, although this convenient synthetic route could be applied for new acyclic scaffold for FIT design.</p> <p>New thymine nucleobase alkyne-amine modifications with various lengths of amine linker (AP-C3 and AP-C6 as phosphoramidites) were developed for the orthogonal labelling of DNA at single thymine sites using amide bond formation and CuAAC click chemistry in a single tube labelling protocol. These novel monomers were investigated in fluorescence resonance energy transfer (FRET) oligonucleotide probes with TO as the FRET donor dye, and thermal duplex melting studies were carried out showing beneficial increase of Tm with TO, where chosen reporter dye caused destabilisation . These new modifications provide an effective platform for highly efficient transfer of energy between donor and receptor fluorophore. However, undesirably high fluorescence was observed in the single stranded probe which could be improved by double incorporation of the AP-C3modification and careful choice of a reporter dye. Thermal melting studies showed modest increase of Tm for unlabelled modification and addition of TO did not results in high additional stabilizing effect. Additionally, this modification could be used as a simple building block for incorporation of a TO intercalator and an additional non-fluorescent label into oligonucleotides.</p> <p>Investigation of the mode of attachment of thiazole orange to oligonucleotides (via its benzothiazole or quinoline moiety), and its point of attachment (nucleobase (major groove) or sugar (minor groove) were explored in several sequence contexts. Obtained results clearly shows preference of major grove (C6, PA) with TO attached via benzothiazole and minor groove (AE) with quinoline attached TO, where best fluorescence properties were achieved for pyrimidines (T and C) as a surrounding bases where purines, especially guanine gives unfavourable fluorescence enhancements. In addition, 2′-OMe sugar modification of thiazole orange probes resulted in a particularly high fluorescent enhancement and very favourable quantum yields on duplex formation. This provides a simple and effective system for detection of DNA and RNA and puts our design on par with more complex systems involving TO, including ECHO probes (exciton-controlled hybridization-sensitive fluorescent oligonucleotide probes, Okamoto); and FIT probes (forced intercalation probes, Seitz). Interestingly, the labelling of amino-modified oligonucleotides with TO-NHS ester is rapid (10 minutes at room temperature), and we were able to label oligonucleotide probes with multiple TO units in almost quantitative yields.</p> |
| spellingShingle | Nucleic acid probes Chemical biology Chemistry RNA Oligonucleotides Klimkowski, P The application of thiazole orange and artificial backbone modifications in the synthesis of fluorescent nucleic acids probes |
| title | The application of thiazole orange and artificial backbone modifications in the synthesis of fluorescent nucleic acids probes |
| title_full | The application of thiazole orange and artificial backbone modifications in the synthesis of fluorescent nucleic acids probes |
| title_fullStr | The application of thiazole orange and artificial backbone modifications in the synthesis of fluorescent nucleic acids probes |
| title_full_unstemmed | The application of thiazole orange and artificial backbone modifications in the synthesis of fluorescent nucleic acids probes |
| title_short | The application of thiazole orange and artificial backbone modifications in the synthesis of fluorescent nucleic acids probes |
| title_sort | application of thiazole orange and artificial backbone modifications in the synthesis of fluorescent nucleic acids probes |
| topic | Nucleic acid probes Chemical biology Chemistry RNA Oligonucleotides |
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