3
views
0
recommends
+1 Recommend
0 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: found
      Is Open Access

      Thermal stabilisation of the short DNA duplexes by acridine-4-carboxamide derivatives

      research-article

      Read this article at

      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          The short oligodeoxynucleotide (ODN) probes are suitable for good discrimination of point mutations. However, the probes suffer from low melting temperatures. In this work, the strategy of using acridine-4-carboxamide intercalators to improve thermal stabilisation is investigated. The study of large series of acridines revealed that optimal stabilisation is achieved upon decoration of acridine by secondary carboxamide carrying sterically not demanding basic function bound through a two-carbon linker. Two highly active intercalators were attached to short probes (13 or 18 bases; designed as a part of HFE gene) by click chemistry into positions 7 and/or 13 and proved to increase the melting temperate ( T m) of the duplex by almost 8°C for the best combination. The acridines interact with both single- and double-stranded DNAs with substantially preferred interaction for the latter. The study of interaction suggested higher affinity of the acridines toward the GC- than AT-rich sequences. Good discrimination of two point mutations was shown in practical application with HFE gene (wild type, H63D C > G and S65C A > C mutations). Acridine itself can also serve as a fluorophore and also allows discrimination of the fully matched sequences from those with point mutations in probes labelled only with acridine.

          Graphical Abstract

          Graphical Abstract

          Acridine-4-carboxamides (both freely in solution and attached covalently to the probe) can increase thermal stability of short oligodeoxynucleotide probes leading to good discrimination of single-based mismatches.

          Related collections

          Most cited references61

          • Record: found
          • Abstract: found
          • Article: not found

          A strain-promoted [3 + 2] azide-alkyne cycloaddition for covalent modification of biomolecules in living systems.

          Selective chemical reactions that are orthogonal to the diverse functionality of biological systems have become important tools in the field of chemical biology. Two notable examples are the Staudinger ligation of azides and phosphines and the Cu(I)-catalyzed [3 + 2] cycloaddition of azides and alkynes ("click chemistry"). The Staudinger ligation has sufficient biocompatibility for performance in living animals but suffers from phosphine oxidation and synthetic challenges. Click chemistry obviates the requirement of phosphines, but the Cu(I) catalyst is toxic to cells, thereby precluding in vivo applications. Here we present a strain-promoted [3 + 2] cycloaddition between cyclooctynes and azides that proceeds under physiological conditions without the need for a catalyst. The utility of the reaction was demonstrated by selective modification of biomolecules in vitro and on living cells, with no apparent toxicity.
            Bookmark
            • Record: found
            • Abstract: found
            • Article: not found

            3'-minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures.

            DNA probes with conjugated minor groove binder (MGB) groups form extremely stable duplexes with single-stranded DNA targets, allowing shorter probes to be used for hybridization based assays. In this paper, sequence specificity of 3'-MGB probes was explored. In comparison with unmodified DNA, MGB probes had higher melting temperature (T(m)) and increased specificity, especially when a mismatch was in the MGB region of the duplex. To exploit these properties, fluorogenic MGB probes were prepared and investigated in the 5'-nuclease PCR assay (real-time PCR assay, TaqMan assay). A 12mer MGB probe had the same T(m)(65 degrees C) as a no-MGB 27mer probe. The fluorogenic MGB probes were more specific for single base mismatches and fluorescence quenching was more efficient, giving increased sensitivity. A/T rich duplexes were stabilized more than G/C rich duplexes, thereby leveling probe T(m)and simplifying design. In summary, MGB probes were more sequence specific than standard DNA probes, especially for single base mismatches at elevated hybridization temperatures.
              Bookmark
              • Record: found
              • Abstract: found
              • Article: not found

              Cu-free click cycloaddition reactions in chemical biology.

              Bioorthogonal chemical reactions are paving the way for new innovations in biology. These reactions possess extreme selectivity and biocompatibility, such that their participating reagents can form covalent bonds within richly functionalized biological systems--in some cases, living organisms. This tutorial review will summarize the history of this emerging field, as well as recent progress in the development and application of bioorthogonal copper-free click cycloaddition reactions.
                Bookmark

                Author and article information

                Contributors
                Journal
                Nucleic Acids Res
                Nucleic Acids Res
                nar
                Nucleic Acids Research
                Oxford University Press
                0305-1048
                1362-4962
                14 October 2022
                26 September 2022
                26 September 2022
                : 50
                : 18
                : 10212-10229
                Affiliations
                Faculty of Pharmacy in Hradec Králové, Charles University , Ak. Heyrovskeho 1203, Hradec Kralove, 500 05, Czech Republic
                Faculty of Pharmacy in Hradec Králové, Charles University , Ak. Heyrovskeho 1203, Hradec Kralove, 500 05, Czech Republic
                Generi Biotech , Machkova 587, Hradec Kralove, 500 11, Czech Republic
                Generi Biotech , Machkova 587, Hradec Kralove, 500 11, Czech Republic
                Generi Biotech , Machkova 587, Hradec Kralove, 500 11, Czech Republic
                Faculty of Pharmacy in Hradec Králové, Charles University , Ak. Heyrovskeho 1203, Hradec Kralove, 500 05, Czech Republic
                Faculty of Pharmacy in Hradec Králové, Charles University , Ak. Heyrovskeho 1203, Hradec Kralove, 500 05, Czech Republic
                Faculty of Pharmacy in Hradec Králové, Charles University , Ak. Heyrovskeho 1203, Hradec Kralove, 500 05, Czech Republic
                Author notes
                To whom correspondence should be addressed. Tel: +420 495067257; Email: zimcik@ 123456faf.cuni.cz
                Correspondence may also be addressed to Filip Kostelansky. Email: kostelaf@ 123456faf.cuni.cz
                Author information
                https://orcid.org/0000-0002-3533-3601
                Article
                gkac777
                10.1093/nar/gkac777
                9561273
                36156152
                d51f1141-dc37-480e-b2e5-224809c7aa7d
                © The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License ( https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@ 123456oup.com

                History
                : 29 August 2022
                : 17 August 2022
                : 09 December 2021
                Page count
                Pages: 18
                Funding
                Funded by: Charles University, DOI 10.13039/100007397;
                Award ID: SVV 260 547
                Award ID: GAUK 994218
                Award ID: PRIMUS/20/SCI/013
                Funded by: European Regional Development Fund, DOI 10.13039/501100008530;
                Award ID: CZ.02.1.01/0.0/0.0/16_019/0000841
                Categories
                AcademicSubjects/SCI00010
                Chemistry

                Genetics
                Genetics

                Comments

                Comment on this article