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28 pages, 3039 KiB

Open AccessArticle

Assembly of Biologically Functional Structures by Nucleic Acid Templating: Implementation of a Strategy to Overcome Inhibition by Template Excess

by Matthew M. Lawler, James T. Kurnick, Leah Fagundes St. Pierre, Estelle E. Newton, Lenora B. Rose and Ian S. Dunn

Molecules 2022, 27(20), 6831; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules27206831 - 12 Oct 2022

Viewed by 1287

Abstract

Delivery of therapeutic molecules to pathogenic cells is often hampered by unintended toxicity to normal cells. In principle, this problem can be circumvented if the therapeutic effector molecule is split into two inactive components, and only assembled on or within the target cell [...] Read more.

Delivery of therapeutic molecules to pathogenic cells is often hampered by unintended toxicity to normal cells. In principle, this problem can be circumvented if the therapeutic effector molecule is split into two inactive components, and only assembled on or within the target cell itself. Such an in situ process can be realized by exploiting target-specific molecules as templates to direct proximity-enhanced assembly. Modified nucleic acids carrying inert precursor fragments can be designed to co-hybridize on a target-specific template nucleic acid, such that the enforced proximity accelerates assembly of a functional molecule for antibody recognition. We demonstrate the in vitro feasibility of this adaptation of nucleic acid-templated synthesis (NATS) using oligonucleotides bearing modified peptides (“haplomers”), for templated assembly of a mimotope recognized by the therapeutic antibody trastuzumab. Enforced proximity promotes mimotope assembly via traceless native chemical ligation. Nevertheless, titration of participating haplomers through template excess is a potential limitation of trimolecular NATS. In order to overcome this problem, we devised a strategy where haplomer hybridization can only occur in the presence of target, without being subject to titration effects. This generalizable NATS modification may find future applications in enabling directed targeting of pathological cells. Full article

(This article belongs to the Collection New Frontiers in Nucleic Acid Chemistry)

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18 pages, 2390 KiB

Open AccessArticle

Oxadiazole/Pyridine-Based Ligands: A Structural Tuning for Enhancing G-Quadruplex Binding

by Filippo Doria, Valentina Pirota, Michele Petenzi, Marie-Paule Teulade-Fichou, Daniela Verga and Mauro Freccero

Molecules 2018, 23(9), 2162; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules23092162 - 28 Aug 2018

Cited by 16 | Viewed by 4241

Abstract

Non-macrocyclic heteroaryls represent a valuable class of ligands for nucleic acid recognition. In this regard, non-macrocyclic pyridyl polyoxazoles and polyoxadiazoles were recently identified as selective G-quadruplex stabilizing compounds with high cytotoxicity and promising anticancer activity. Herein, we describe the synthesis of a new [...] Read more.

Non-macrocyclic heteroaryls represent a valuable class of ligands for nucleic acid recognition. In this regard, non-macrocyclic pyridyl polyoxazoles and polyoxadiazoles were recently identified as selective G-quadruplex stabilizing compounds with high cytotoxicity and promising anticancer activity. Herein, we describe the synthesis of a new family of heteroaryls containing oxadiazole and pyridine moieties targeting DNA G-quadruplexes. To perform a structure–activity analysis identifying determinants of activity and selectivity, we followed a convergent synthetic pathway to modulate the nature and number of the heterocycles (1,3-oxazole vs. 1,2,4-oxadiazole and pyridine vs. benzene). Each ligand was evaluated towards secondary nucleic acid structures, which have been chosen as a prototype to mimic cancer-associated G-quadruplex structures (e.g., the human telomeric sequence, c-myc and c-kit promoters). Interestingly, heptapyridyl-oxadiazole compounds showed preferential binding towards the telomeric sequence (22AG) in competitive conditions vs. duplex DNA. In addition, G4-FID assays suggest a different binding mode from the classical stacking on the external G-quartet. Additionally, CD titrations in the presence of the two most promising compounds for affinity, TOxAzaPy and TOxAzaPhen, display a structural transition of 22AG in K-rich buffer. This investigation suggests that the pyridyl-oxadiazole motif is a promising recognition element for G-quadruplexes, combining seven heteroaryls in a single binding unit. Full article

(This article belongs to the Collection New Frontiers in Nucleic Acid Chemistry)

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18 pages, 2275 KiB

Open AccessArticle

A Fragment-Based Approach for the Development of G-Quadruplex Ligands: Role of the Amidoxime Moiety

by Martina Tassinari, Alberto Lena, Elena Butovskaya, Valentina Pirota, Matteo Nadai, Mauro Freccero, Filippo Doria and Sara N. Richter

Molecules 2018, 23(8), 1874; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules23081874 - 27 Jul 2018

Cited by 11 | Viewed by 5013

Abstract

G-quadruplex (G4) nucleic acid structures have been reported to be involved in several human pathologies, including cancer, neurodegenerative disorders and infectious diseases; however, G4 targeting compounds still need implementation in terms of drug-like properties and selectivity in order to reach the clinical use. [...] Read more.

G-quadruplex (G4) nucleic acid structures have been reported to be involved in several human pathologies, including cancer, neurodegenerative disorders and infectious diseases; however, G4 targeting compounds still need implementation in terms of drug-like properties and selectivity in order to reach the clinical use. So far, G4 ligands have been mainly identified through high-throughput screening methods or design of molecules with pre-set features. Here, we describe the development of new heterocyclic ligands through a fragment-based drug discovery (FBDD) approach. The ligands were designed against the major G4 present in the long terminal repeat (LTR) promoter region of the human immunodeficiency virus-1 (HIV-1), the stabilization of which has been shown to suppress viral gene expression and replication. Our method is based on the generation of molecular fragment small libraries, screened against the target to further elaborate them into lead compounds. We screened 150 small molecules, composed by structurally and chemically different fragments, selected from commercially available and in-house compounds; synthetic elaboration yielded several G4 ligands and two final G4 binders, both embedding an amidoxime moiety; one of these two compounds showed preferential binding for the HIV-1 LTR G4. This work presents the discovery of a novel potential pharmacophore and highlights the possibility to apply a fragment-based approach to develop G4 ligands with unexpected chemical features. Full article

(This article belongs to the Collection New Frontiers in Nucleic Acid Chemistry)

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12 pages, 2670 KiB

Open AccessArticle

Linking Temperature, Cation Concentration and Water Activity for the B to Z Conformational Transition in DNA

by Jaime M. Ferreira and Richard D. Sheardy

Molecules 2018, 23(7), 1806; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules23071806 - 21 Jul 2018

Cited by 7 | Viewed by 4288

Abstract

High concentrations of Na⁺ or [Co(NH₃)₆]³⁺ can induce the B to Z conformational transition in alternating (dC-dG) oligo and polynucleotides. The use of short DNA oligomers (dC-dG)₄ and (dm⁵C-dG)₄ as models can allow [...] Read more.

High concentrations of Na⁺ or [Co(NH₃)₆]³⁺ can induce the B to Z conformational transition in alternating (dC-dG) oligo and polynucleotides. The use of short DNA oligomers (dC-dG)₄ and (dm⁵C-dG)₄ as models can allow a thermodynamic characterization of the transition. Both form right handed double helical structures (B-DNA) in standard phosphate buffer with 115 mM Na⁺ at 25 °C. However, at 2.0 M Na⁺ or 200 μM [Co(NH₃)₆]³⁺, (dm⁵C-dG)₄ assumes a left handed double helical structure (Z-DNA) while the unmethylated (dC-dG)₄ analogue remains right handed under those conditions. We have previously demonstrated that the enthalpy of the transition at 25 °C for either inducer can be determined using isothermal titration calorimetry (ITC). Here, ITC is used to investigate the linkages between temperature, water activity and DNA conformation. We found that the determined enthalpy for each titration varied linearly with temperature allowing determination of the heat capacity change (ΔC_p) between the initial and final states. As expected, the ΔC_p values were dependent upon the cation (i.e., Na⁺ vs. [Co(NH₃)₆]³⁺) as well as the sequence of the DNA oligomer (i.e., methylated vs. unmethylated). Osmotic stress experiments were carried out to determine the gain or loss of water by the oligomer induced by the titration. The results are discussed in terms of solvent accessible surface areas, electrostatic interactions and the role of water. Full article

(This article belongs to the Collection New Frontiers in Nucleic Acid Chemistry)

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5333 KiB

Open AccessArticle

Binding of Harmine Derivatives to DNA: A Spectroscopic Investigation

by Bruno Pagano, Marco Caterino, Rosanna Filosa and Concetta Giancola

Molecules 2017, 22(11), 1831; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules22111831 - 27 Oct 2017

Cited by 12 | Viewed by 5144

Abstract

Harmine belongs to a group of β-carboline alkaloids endowed with antitumor properties. Harmine and its derivatives are thought to bind to DNA and interfere with topoisomerase activities. We investigated the base-dependent binding of harmine, and three of its synthetic anticancer-active derivatives to the [...] Read more.

Harmine belongs to a group of β-carboline alkaloids endowed with antitumor properties. Harmine and its derivatives are thought to bind to DNA and interfere with topoisomerase activities. We investigated the base-dependent binding of harmine, and three of its synthetic anticancer-active derivatives to the genomic DNA from calf thymus and two synthetic 20-mer double helices, the poly(dG-dC)·poly(dG-dC) and the poly(dA-dT)·poly(dA-dT), by means of UV-Vis and circular dichroism (CD) spectroscopies. The data show that the DNA binding and stabilising properties of the investigated derivatives are base pair-dependent. These results could be used as a guide to design and develop further bioactive analogues. Full article

(This article belongs to the Collection New Frontiers in Nucleic Acid Chemistry)

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2236 KiB

Open AccessArticle

The Molecular Effect of Diagnostic Absorbed Doses from ¹³¹I on Papillary Thyroid Cancer Cells In Vitro

by Mariusz Stasiołek, Zbigniew Adamczewski, Przemysław W. Śliwka, Bartosz Puła, Bolesław Karwowski, Anna Merecz-Sadowska, Marek Dedecjus and Andrzej Lewiński

Molecules 2017, 22(6), 993; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules22060993 - 15 Jun 2017

Cited by 5 | Viewed by 4541

Abstract

Diagnostic whole-body scan is a standard procedure in patients with thyroid cancer prior to the application of a therapeutic dose of ¹³¹I. Unfortunately, administration of the radioisotope in a diagnostic dose may decrease further radioiodine uptake—the phenomenon called “thyroid stunning”. We estimated [...] Read more.

Diagnostic whole-body scan is a standard procedure in patients with thyroid cancer prior to the application of a therapeutic dose of ¹³¹I. Unfortunately, administration of the radioisotope in a diagnostic dose may decrease further radioiodine uptake—the phenomenon called “thyroid stunning”. We estimated radiation absorbed dose-dependent changes in genetic material, in particular in the sodium iodide symporter (NIS) gene promoter, and the NIS protein level in a K1 cell line derived from the metastasis of a human papillary thyroid carcinoma exposed to ¹³¹I in culture. The different activities applied were calculated to result in absorbed doses of 5, 10 and 20 Gy. Radioiodine did not affect the expression of the NIS gene at the mRNA level, however, we observed significant changes in the NIS protein level in K1 cells. The decrease of the NIS protein level observed in the cells subjected to the lowest absorbed dose was paralleled by a significant increase in 8-oxo-dG concentrations (p < 0.01) and followed by late activation of the DNA repair pathways. Our findings suggest that the impact of ¹³¹I radiation on thyroid cells, in the range compared to doses absorbed during diagnostic procedures, is not linear and depends on various factors including the cellular components of thyroid pathology. Full article

(This article belongs to the Collection New Frontiers in Nucleic Acid Chemistry)

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2934 KiB

Open AccessArticle

Synthesis and Biological Evaluation of Triazolyl 13α-Estrone–Nucleoside Bioconjugates

by Brigitta Bodnár, Erzsébet Mernyák, János Wölfling, Gyula Schneider, Bianka Edina Herman, Mihály Szécsi, Izabella Sinka, István Zupkó, Zoltán Kupihár and Lajos Kovács

Molecules 2016, 21(9), 1212; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules21091212 - 10 Sep 2016

Cited by 16 | Viewed by 7092

Abstract

2′-Deoxynucleoside conjugates of 13α-estrone were synthesized by applying the copper-catalyzed alkyne–azide click reaction (CuAAC). For the introduction of the azido group the 5′-position of the nucleosides and a propargyl ether functional group on the 3-hydroxy group of 13α-estrone were chosen. The best yields [...] Read more.

2′-Deoxynucleoside conjugates of 13α-estrone were synthesized by applying the copper-catalyzed alkyne–azide click reaction (CuAAC). For the introduction of the azido group the 5′-position of the nucleosides and a propargyl ether functional group on the 3-hydroxy group of 13α-estrone were chosen. The best yields were realized in our hands when the 3′-hydroxy groups of the nucleosides were protected by acetyl groups and the 5′-hydroxy groups were modified by the tosyl–azide exchange method. The commonly used conditions for click reaction between the protected-5′-azidonucleosides and the steroid alkyne was slightly modified by using 1.5 equivalent of Cu(I) catalyst. All the prepared conjugates were evaluated in vitro by means of MTT assays for antiproliferative activity against a panel of human adherent cell lines (HeLa, MCF-7 and A2780) and the potential inhibitory activity of the new conjugates on human 17β-hydroxysteroid dehydrogenase 1 (17β-HSD1) was investigated via in vitro radiosubstrate incubation. Some protected conjugates displayed moderate antiproliferative properties against a panel of human adherent cancer cell lines (the protected cytidine conjugate proved to be the most potent with IC₅₀ value of 9 μM). The thymidine conjugate displayed considerable 17β-HSD1 inhibitory activity (IC₅₀ = 19 μM). Full article

(This article belongs to the Collection New Frontiers in Nucleic Acid Chemistry)

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1251 KiB

Open AccessArticle

DNA Three Way Junction Core Decorated with Amino Acids-Like Residues-Synthesis and Characterization

by Claudia Addamiano, Béatrice Gerland, Corinne Payrastre and Jean-Marc Escudier

Molecules 2016, 21(9), 1082; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules21091082 - 23 Aug 2016

Cited by 4 | Viewed by 7471

Abstract

Construction and physico-chemical behavior of DNA three way junction (3WJ) functionalized by protein-like residues (imidazole, alcohol and carboxylic acid) at unpaired positions at the core is described. One 5′-C(S)-propargyl-thymidine nucleotide was specifically incorporated on each strand to react through a post [...] Read more.

Construction and physico-chemical behavior of DNA three way junction (3WJ) functionalized by protein-like residues (imidazole, alcohol and carboxylic acid) at unpaired positions at the core is described. One 5′-C(S)-propargyl-thymidine nucleotide was specifically incorporated on each strand to react through a post synthetic CuACC reaction with either protected imidazolyl-, hydroxyl- or carboxyl-azide. Structural impacts of 5′-C(S)-functionalization were investigated to evaluate how 3WJ flexibility/stability is affected. Full article

(This article belongs to the Collection New Frontiers in Nucleic Acid Chemistry)

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2286 KiB

Open AccessArticle

Chemical Incorporation of Chain-Terminating Nucleoside Analogs as 3′-Blocking DNA Damage and Their Removal by Human ERCC1-XPF Endonuclease

by Junpei Yamamoto, Chiaki Takahata, Isao Kuraoka, Kouji Hirota and Shigenori Iwai

Molecules 2016, 21(6), 766; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules21060766 - 11 Jun 2016

Cited by 3 | Viewed by 7097

Abstract

Nucleoside/nucleotide analogs that lack the 3′-hydroxy group are widely utilized for HIV therapy. These chain-terminating nucleoside analogs (CTNAs) block DNA synthesis after their incorporation into growing DNA, leading to the antiviral effects. However, they are also considered to be DNA damaging agents, and [...] Read more.

Nucleoside/nucleotide analogs that lack the 3′-hydroxy group are widely utilized for HIV therapy. These chain-terminating nucleoside analogs (CTNAs) block DNA synthesis after their incorporation into growing DNA, leading to the antiviral effects. However, they are also considered to be DNA damaging agents, and tyrosyl-DNA phosphodiesterase 1, a DNA repair enzyme, is reportedly able to remove such CTNA-modifications of DNA. Here, we have synthesized phosphoramidite building blocks of representative CTNAs, such as acyclovir, abacavir, carbovir, and lamivudine, and oligonucleotides with the 3′-CTNAs were successfully synthesized on solid supports. Using the chemically synthesized oligonucleotides, we investigated the excision of the 3′-CTNAs in DNA by the human excision repair cross complementing protein 1-xeroderma pigmentosum group F (ERCC1-XPF) endonuclease, which is one of the main components of the nucleotide excision repair pathway. A biochemical analysis demonstrated that the ERCC1-XPF endonuclease cleaved 2–7 nt upstream from the 3′-blocking CTNAs, and that DNA synthesis by the Klenow fragment was resumed after the removal of the CTNAs, suggesting that ERCC1-XPF participates in the repair of the CTNA-induced DNA damage. Full article

(This article belongs to the Collection New Frontiers in Nucleic Acid Chemistry)

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4301 KiB

Open AccessArticle

DNA Scaffolded Silver Clusters: A Critical Study

by Bidisha Sengupta, Christa Corley, Keith Cobb, Anthony Saracino and Steffen Jockusch

Molecules 2016, 21(2), 216; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules21020216 - 17 Feb 2016

Cited by 13 | Viewed by 5423

Abstract

Fluorescent silver nanoclusters (Ag-NCs) are in prominence as novel sensing materials due to their biocompatibility, photostability, and molecule-like optical properties. The present work is carried out on an array (17 sequences) of 16 bases long cytosine rich, single stranded DNA templates 5′-C₃ [...] Read more.

Fluorescent silver nanoclusters (Ag-NCs) are in prominence as novel sensing materials due to their biocompatibility, photostability, and molecule-like optical properties. The present work is carried out on an array (17 sequences) of 16 bases long cytosine rich, single stranded DNA templates 5′-C₃X_iC₃X_iiC₃X_iiiC₃X_iv-3′ where i, ii, iii, iv correspond to T/G/C deoxynucleobases (with default base A). Among all the oligonucleotides, a sequence C₃AC₃AC₃TC₃G (3T4G) has been identified, which grows three different near-infrared-emitting NC species with absorption/emission maxima at ~620/700 (species I), 730/800 (species II), and 830 (Species III) nm, respectively. The nature of the spectral profiles, along with relevant parameters namely absorption maximum (\(\lambda_{abs}^{max}\)), emission maximum (\(\lambda_{em}^{max}\)), anisotropy (r), lifetime (\(\tau\)), circular dichroism spectral data are used to understand the microenvironments of the fluorescent NC species I, II, and III. DNA:Ag stiochiometric, pH and solvent dependent studies proved that i-motif scaffolds with different folding topologies are associated with the growth of these three species and a certain concentration of silver and H⁺ favor the growth of species III. Size exclusion chromatographic measurements provided similar indications that a folded, more compact, classic i-motif template is associated with the formation of the longer NIR (~830 nm) absorbing species. This study provides a more definitive approach to design and obtain a targeted DNA templated Ag-NC with required emission properties for biophysical and cellular applications. Full article

(This article belongs to the Special Issue Frontiers in Nucleic Acid Chemistry)

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3630 KiB

Open AccessArticle

A 7-Deazaadenosylaziridine Cofactor for Sequence-Specific Labeling of DNA by the DNA Cytosine-C5 Methyltransferase M.HhaI

by Falk Kunkel, Rudi Lurz and Elmar Weinhold

Molecules 2015, 20(11), 20805-20822; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules201119723 - 23 Nov 2015

Cited by 12 | Viewed by 6580

Abstract

DNA methyltransferases (MTases) catalyze the transfer of the activated methyl group of the cofactor S-adenosyl-l-methionine (AdoMet or SAM) to the exocyclic amino groups of adenine or cytosine or the C5 ring atom of cytosine within specific DNA sequences. The DNA adenine-N6 MTase [...] Read more.

DNA methyltransferases (MTases) catalyze the transfer of the activated methyl group of the cofactor S-adenosyl-l-methionine (AdoMet or SAM) to the exocyclic amino groups of adenine or cytosine or the C5 ring atom of cytosine within specific DNA sequences. The DNA adenine-N6 MTase from Thermus aquaticus (M.TaqI) is also capable of coupling synthetic N-adenosylaziridine cofactor analogues to its target adenine within the double-stranded 5′-TCGA-3′ sequence. This M.TaqI-mediated coupling reaction was exploited to sequence-specifically deliver fluorophores and biotin to DNA using N-adenosylaziridine derivatives carrying reporter groups at the 8-position of the adenine ring. However, these 8-modified aziridine cofactors were poor substrates for the DNA cytosine-C5 MTase from Haemophilus haemolyticus (M.HhaI). Based on the crystal structure of M.HhaI in complex with a duplex oligodeoxynucleotide and the cofactor product, we synthesized a stable 7-deazaadenosylaziridine derivative with a biotin group attached to the 7-position via a flexible linker. This 7-modified aziridine cofactor can be efficiently used by M.HhaI for the direct, quantitative and sequence-specific delivery of biotin to the second cytosine within 5′-GCGC-3′ sequences in short duplex oligodeoxynucleotides and plasmid DNA. In addition, we demonstrate that biotinylation by M.HhaI depends on the methylation status of the target cytosine and, thus, could provide a method for cytosine-C5 DNA methylation detection in mammalian DNA. Full article

(This article belongs to the Special Issue Frontiers in Nucleic Acid Chemistry)

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968 KiB

Open AccessArticle

Synthesis, Hydrolysis, and Protonation-Promoted Intramolecular Reductive Breakdown of Potential NRTIs: Stavudine α-P-Borano-γ-P-N-l-tryptophanyltriphosphates

by Zhihong Xu and Barbara Ramsay Shaw

Molecules 2015, 20(10), 18808-18826; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules201018808 - 16 Oct 2015

Cited by 2 | Viewed by 6879

Abstract

Phosphorus-modified prodrugs of dideoxynucleoside triphosphates (ddNTPs) have shown promise as pronucleotide strategies for improving antiviral activity compared to their parent dideoxynucleosides. Borane modified NTPs offer a promising choice as nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs). However, the availability of α-P-borano-γ-P-substituted [...] Read more.

Phosphorus-modified prodrugs of dideoxynucleoside triphosphates (ddNTPs) have shown promise as pronucleotide strategies for improving antiviral activity compared to their parent dideoxynucleosides. Borane modified NTPs offer a promising choice as nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs). However, the availability of α-P-borano-γ-P-substituted NTP analogs remains limited due to challenges with synthesis and purification. Here, we report the chemical synthesis and stability of a new potential class of NRTI prodrugs: stavudine (d4T) 5′-α-P-borano-γ-P-N-L-tryptophanyltriphosphates. One-pot synthesis of these compounds was achieved via a modified cyclic trimetaphosphate approach. Pure Rp and Sp diastereomers were obtained after HPLC separation. Based on LC-MS analysis, we report degradation pathways, half-lives (5–36 days) and mechanisms arising from structural differences to generate the corresponding borano tri- and di-phosphates, and H-phosphonate, via several parallel routes in buffer at physiologically relevant pH and temperature. Here, the major hydrolysis products, d4T α-P-boranotriphosphate Rp and Sp isomers, were isolated by HPLC and identified with spectral data. We first propose that one of the major degradation products, d4T H-phosphonate, was generated from the d4T pronucleotides via a protonation-promoted intramolecular reduction followed by a second step nucleophilic attack. This report could provide valuable information for pronucleotide-based drug design in terms of selective release of target nucleotides. Full article

(This article belongs to the Special Issue Frontiers in Nucleic Acid Chemistry)

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2450 KiB

Open AccessArticle

Comparative Incorporation of PNA into DNA Nanostructures

by Ronnie O. Pedersen, Jing Kong, Catalina Achim and Thomas H. LaBean

Molecules 2015, 20(9), 17645-17658; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules200917645 - 23 Sep 2015

Cited by 13 | Viewed by 6605

Abstract

DNA has shown great promise as a building material for self-assembling nanoscale structures. To further develop the potential of this technology, more methods are needed for functionalizing DNA-based nanostructures to increase their chemical diversity. Peptide nucleic acid (PNA) holds great promise for realizing [...] Read more.

DNA has shown great promise as a building material for self-assembling nanoscale structures. To further develop the potential of this technology, more methods are needed for functionalizing DNA-based nanostructures to increase their chemical diversity. Peptide nucleic acid (PNA) holds great promise for realizing this goal, as it conveniently allows for inclusion of both amino acids and peptides in nucleic acid-based structures. In this work, we explored incorporation of a positively charged PNA within DNA nanostructures. We investigated the efficiency of annealing a lysine-containing PNA probe with complementary, single-stranded DNA sequences within nanostructures, as well as the efficiency of duplex invasion and its dependence on salt concentration. Our results show that PNA allows for toehold-free strand displacement and that incorporation yield depends critically on binding site geometry. These results provide guidance for the design of PNA binding sites on nucleic acid nanostructures with an eye towards optimizing fabrication yield. Full article

(This article belongs to the Special Issue Frontiers in Nucleic Acid Chemistry)

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2299 KiB

Open AccessArticle

Synthesis and Evaluation of a Rationally Designed Click-Based Library for G-Quadruplex Selective DNA Photocleavage

by Dominic McBrayer and Sean M. Kerwin

Molecules 2015, 20(9), 16446-16465; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules200916446 - 10 Sep 2015

Cited by 2 | Viewed by 5682

Abstract

DNA containing repeating G-rich sequences can adopt higher-order structures known as G-quadruplexes (G4). These structures are believed to form within telomeres and the promoter regions of some genes, particularly in a number of proto-oncogenes, where they may play a role in regulating transcription. [...] Read more.

DNA containing repeating G-rich sequences can adopt higher-order structures known as G-quadruplexes (G4). These structures are believed to form within telomeres and the promoter regions of some genes, particularly in a number of proto-oncogenes, where they may play a role in regulating transcription. Alternatively, G4 DNA may act as a barrier to replication. To investigate these potential biological roles, probes that combine highly selective G4 DNA targeting with photocleavage activity can allow temporal detection of G4 DNA, providing opportunities to obtain novel insights about the biological roles of G4 DNA. We have designed, synthesized, and screened a small library of potential selective G-quadruplex DNA photocleavage agents incorporating the G-quadruplex targeting moiety of 360A with known photocleavage groups linked via “click” chemistry. Full article

(This article belongs to the Special Issue Frontiers in Nucleic Acid Chemistry)

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Open AccessArticle

Mixed-Sequence Recognition of Double-Stranded DNA Using Enzymatically Stable Phosphorothioate Invader Probes

by Brooke A. Anderson, Saswata Karmakar and Patrick J. Hrdlicka

Molecules 2015, 20(8), 13780-13793; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules200813780 - 29 Jul 2015

Cited by 7 | Viewed by 5870

Abstract

Development of probes that allow for sequence-unrestricted recognition of double-stranded DNA (dsDNA) continues to attract much attention due to the prospect for molecular tools that enable detection, regulation, and manipulation of genes. We have recently introduced so-called Invader probes as alternatives to more [...] Read more.

Development of probes that allow for sequence-unrestricted recognition of double-stranded DNA (dsDNA) continues to attract much attention due to the prospect for molecular tools that enable detection, regulation, and manipulation of genes. We have recently introduced so-called Invader probes as alternatives to more established approaches such as triplex-forming oligonucleotides, peptide nucleic acids and polyamides. These short DNA duplexes are activated for dsDNA recognition by installment of +1 interstrand zippers of intercalator-functionalized nucleotides such as 2′-N-(pyren-1-yl)methyl-2′-N-methyl-2′-aminouridine and 2′-O-(pyren-1-yl)methyluridine, which results in violation of the nearest neighbor exclusion principle and duplex destabilization. The individual probes strands have high affinity toward complementary DNA strands, which generates the driving force for recognition of mixed-sequence dsDNA regions. In the present article, we characterize Invader probes that are based on phosphorothioate backbones (PS-DNA Invaders). The change from the regular phosphodiester backbone furnishes Invader probes that are much more stable to nucleolytic degradation, while displaying acceptable dsDNA-recognition efficiency. PS-DNA Invader probes therefore present themselves as interesting probes for dsDNA-targeting applications in cellular environments and living organisms. Full article

(This article belongs to the Special Issue Frontiers in Nucleic Acid Chemistry)

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1128 KiB

Open AccessCommunication

Synthesis and Enzymatic Incorporation of Modified Deoxyuridine Triphosphates

by Erkai Liu, Curtis H. Lam and David M. Perrin

Molecules 2015, 20(8), 13591-13602; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules200813591 - 24 Jul 2015

Cited by 11 | Viewed by 6091

Abstract

To expand the chemical functionality of DNAzymes and aptamers, several new modified deoxyuridine triphosphates have been synthesized. An important precursor that enables this aim is 5-aminomethyl dUTP, whereby the pendent amine serves as a handle for further synthetic functionalization. Five functional groups were [...] Read more.

To expand the chemical functionality of DNAzymes and aptamers, several new modified deoxyuridine triphosphates have been synthesized. An important precursor that enables this aim is 5-aminomethyl dUTP, whereby the pendent amine serves as a handle for further synthetic functionalization. Five functional groups were conjugated to 5-aminomethyl dUTP. Incorporation assays were performed on several templates that demand 2–5 sequential incorporation events using several commercially available DNA polymerases. It was found that Vent (exo-) DNA polymerase efficiently incorporates all five modified dUTPs. In addition, all nucleoside triphosphates were capable of supporting a double-stranded exponential PCR amplification. Modified PCR amplicons were PCR amplified into unmodified DNA and sequenced to verify that genetic information was conserved through incorporation, amplification, and reamplification. Overall these modified dUTPs represent new candidate substrates for use in selections using modified nucleotide libraries. Full article

(This article belongs to the Special Issue Frontiers in Nucleic Acid Chemistry)

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1546 KiB

Open AccessArticle

The Influence of the Terminal Phosphorothioate Diester Bond on the DNA Oxidation Process. An Experimental and Theoretical Approach

by Boleslaw T. Karwowski

Molecules 2015, 20(7), 12400-12411; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules200712400 - 08 Jul 2015

Cited by 2 | Viewed by 5553

Abstract

In this study, the influence of the terminal phosphorothioate (PT) internucleotide bond in ds-DNA on the oxidation process was taken into consideration. The interaction of UV with the targeted oligonucleotide leads to an electron ejection and radical cation “hole” migration through the [...] Read more.

In this study, the influence of the terminal phosphorothioate (PT) internucleotide bond in ds-DNA on the oxidation process was taken into consideration. The interaction of UV with the targeted oligonucleotide leads to an electron ejection and radical cation “hole” migration through the ds-DNA until it is trapped irreversibly in a suitable place. Phosphorothiate internucleotide bonds were detected in the bacterial genome; however, their role is still unclear. In this study a PAGE analysis of irradiated ds-DNA showed that the degradation rea ction was slowed down by the presence PT next to the anthraquinone moiety. Further, theoretical study shows that [R_P] AQ-PS-dG can adopt a slightly lower ionisation potential energy and triplet excited state with a subsequent slightly higher adiabatic electron affinity value in comparison with [S_P] AQ-PS-dG and AQ-PO-dG. Moreover, the energy gap between HOMO and LUMO, indicated the radical stabilisation properties of [R_P] AQ-PS-dG, which can hinder the charge transfer through ds-DNA. Full article

(This article belongs to the Special Issue Frontiers in Nucleic Acid Chemistry)

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Open AccessArticle

Modulation of the RNA Interference Activity Using Central Mismatched siRNAs and Acyclic Threoninol Nucleic Acids (aTNA) Units

by Adele Alagia, Montserrat Terrazas and Ramon Eritja

Molecules 2015, 20(5), 7602-7619; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules20057602 - 24 Apr 2015

Cited by 15 | Viewed by 7469

Abstract

The understanding of the mechanisms behind nucleotide recognition by Argonaute 2, core protein of the RNA-induced silencing complex, is a key aspect in the optimization of small interfering RNAs (siRNAs) activity. To date, great efforts have been focused on the modification of certain [...] Read more.

The understanding of the mechanisms behind nucleotide recognition by Argonaute 2, core protein of the RNA-induced silencing complex, is a key aspect in the optimization of small interfering RNAs (siRNAs) activity. To date, great efforts have been focused on the modification of certain regions of siRNA, such as the 3'/5'-termini and the seed region. Only a few reports have described the roles of central positions flanking the cleavage site during the silence process. In this study, we investigate the potential correlations between the thermodynamic and silencing properties of siRNA molecules carrying, at internal positions, an acyclic L-threoninol nucleic acid (aTNA) modification. Depending on position, the silencing is weakened or impaired. Furthermore, we evaluate the contribution of mismatches facing either a natural nucleotide or an aTNA modification to the siRNA potency. The position 11 of the antisense strand is more permissive to mismatches and aTNA modification, in respect to the position 10. Additionally, comparing the ON-/OFF-target silencing of central mismatched siRNAs with 5'-terminal modified siRNA, we concluded: (i) central perturbation of duplex pairing features weights more on potency rather than silencing asymmetry; (ii) complete bias for the ON-target silencing can be achieved with single L-threoninol modification near the 5'-end of the sense strand. Full article

(This article belongs to the Special Issue Frontiers in Nucleic Acid Chemistry)

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18 pages, 1283 KiB

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Determining RNA Natural Modifications and Nucleoside Analog-Labeled Sites by a Chemical/Enzyme-Induced Base Mutation Principle

by Ziming Bao, Tengwei Li and Jianzhao Liu

Molecules 2023, 28(4), 1517; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules28041517 - 04 Feb 2023

Cited by 3 | Viewed by 1966

Abstract

The natural chemical modifications of messenger RNA (mRNA) in living organisms have shown essential roles in both physiology and pathology. The mapping of mRNA modifications is critical for interpreting their biological functions. In another dimension, the synthesized nucleoside analogs can enable chemical labeling [...] Read more.

The natural chemical modifications of messenger RNA (mRNA) in living organisms have shown essential roles in both physiology and pathology. The mapping of mRNA modifications is critical for interpreting their biological functions. In another dimension, the synthesized nucleoside analogs can enable chemical labeling of cellular mRNA through a metabolic pathway, which facilitates the study of RNA dynamics in a pulse-chase manner. In this regard, the sequencing tools for mapping both natural modifications and nucleoside tags on mRNA at single base resolution are highly necessary. In this work, we review the progress of chemical sequencing technology for determining both a variety of naturally occurring base modifications mainly on mRNA and a few on transfer RNA and metabolically incorporated artificial base analogs on mRNA, and further discuss the problems and prospects in the field. Full article

(This article belongs to the Collection New Frontiers in Nucleic Acid Chemistry)

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19 pages, 3287 KiB

Open AccessReview

Single-Labeled Oligonucleotides Showing Fluorescence Changes upon Hybridization with Target Nucleic Acids

by Gil Tae Hwang

Molecules 2018, 23(1), 124; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules23010124 - 08 Jan 2018

Cited by 23 | Viewed by 7973

Abstract

Sequence-specific detection of nucleic acids has been intensively studied in the field of molecular diagnostics. In particular, the detection and analysis of single-nucleotide polymorphisms (SNPs) is crucial for the identification of disease-causing genes and diagnosis of diseases. Sequence-specific hybridization probes, such as molecular [...] Read more.

Sequence-specific detection of nucleic acids has been intensively studied in the field of molecular diagnostics. In particular, the detection and analysis of single-nucleotide polymorphisms (SNPs) is crucial for the identification of disease-causing genes and diagnosis of diseases. Sequence-specific hybridization probes, such as molecular beacons bearing the fluorophore and quencher at both ends of the stem, have been developed to enable DNA mutation detection. Interestingly, DNA mutations can be detected using fluorescently labeled oligonucleotide probes with only one fluorophore. This review summarizes recent research on single-labeled oligonucleotide probes that exhibit fluorescence changes after encountering target nucleic acids, such as guanine-quenching probes, cyanine-containing probes, probes containing a fluorophore-labeled base, and microenvironment-sensitive probes. Full article

(This article belongs to the Collection New Frontiers in Nucleic Acid Chemistry)

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Recent Advances in Nucleic Acid Targeting Probes and Supramolecular Constructs Based on Pyrene-Modified Oligonucleotides

by Olga A. Krasheninina, Darya S. Novopashina, Evgeny K. Apartsin and Alya G. Venyaminova

Molecules 2017, 22(12), 2108; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules22122108 - 30 Nov 2017

Cited by 36 | Viewed by 9800

Abstract

In this review, we summarize the recent advances in the use of pyrene-modified oligonucleotides as a platform for functional nucleic acid-based constructs. Pyrene is of special interest for the development of nucleic acid-based tools due to its unique fluorescent properties (sensitivity of fluorescence [...] Read more.

In this review, we summarize the recent advances in the use of pyrene-modified oligonucleotides as a platform for functional nucleic acid-based constructs. Pyrene is of special interest for the development of nucleic acid-based tools due to its unique fluorescent properties (sensitivity of fluorescence to the microenvironment, ability to form excimers and exciplexes, long fluorescence lifetime, high quantum yield), ability to intercalate into the nucleic acid duplex, to act as a π-π-stacking (including anchoring) moiety, and others. These properties of pyrene have been used to construct novel sensitive fluorescent probes for the sequence-specific detection of nucleic acids and the discrimination of single nucleotide polymorphisms (SNPs), aptamer-based biosensors, agents for binding of double-stranded DNAs, and building blocks for supramolecular complexes. Special attention is paid to the influence of the design of pyrene-modified oligonucleotides on their properties, i.e., the structure-function relationships. The perspectives for the applications of pyrene-modified oligonucleotides in biomolecular studies, diagnostics, and nanotechnology are discussed. Full article

(This article belongs to the Collection New Frontiers in Nucleic Acid Chemistry)

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Evolution of Complex Target SELEX to Identify Aptamers against Mammalian Cell-Surface Antigens

by Prabodhika Mallikaratchy

Molecules 2017, 22(2), 215; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules22020215 - 30 Jan 2017

Cited by 74 | Viewed by 9467

Abstract

The demand has increased for sophisticated molecular tools with improved detection limits. Such molecules should be simple in structure, yet stable enough for clinical applications. Nucleic acid aptamers (NAAs) represent a class of molecules able to meet this demand. In particular, aptamers, a [...] Read more.

The demand has increased for sophisticated molecular tools with improved detection limits. Such molecules should be simple in structure, yet stable enough for clinical applications. Nucleic acid aptamers (NAAs) represent a class of molecules able to meet this demand. In particular, aptamers, a class of small nucleic acid ligands that are composed of single-stranded modified/unmodified RNA/DNA molecules, can be evolved from a complex library using Systematic Evolution of Ligands by EXponential enrichment (SELEX) against almost any molecule. Since its introduction in 1990, in stages, SELEX technology has itself undergone several modifications, improving selection and broadening the repertoire of targets. This review summarizes these milestones that have pushed the field forward, allowing researchers to generate aptamers that can potentially be applied as therapeutic and diagnostic agents. Full article

(This article belongs to the Collection New Frontiers in Nucleic Acid Chemistry)

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Therapeutic Oligonucleotides Targeting Liver Disease: TTR Amyloidosis

by Christoph Niemietz, Gursimran Chandhok and Hartmut Schmidt

Molecules 2015, 20(10), 17944-17975; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules201017944 - 30 Sep 2015

Cited by 24 | Viewed by 13237

Abstract

The liver has become an increasingly interesting target for oligonucleotide therapy. Mutations of the gene encoding transthyretin (TTR), expressed in vast amounts by the liver, result in a complex degenerative disease, termed familial amyloid polyneuropathy (FAP). Misfolded variants of TTR are linked to [...] Read more.

The liver has become an increasingly interesting target for oligonucleotide therapy. Mutations of the gene encoding transthyretin (TTR), expressed in vast amounts by the liver, result in a complex degenerative disease, termed familial amyloid polyneuropathy (FAP). Misfolded variants of TTR are linked to the establishment of extracellular protein deposition in various tissues, including the heart and the peripheral nervous system. Recent progress in the chemistry and formulation of antisense (ASO) and small interfering RNA (siRNA) designed for a knockdown of TTR mRNA in the liver has allowed to address the issue of gene-specific molecular therapy in a clinical setting of FAP. The two therapeutic oligonucleotides bind to RNA in a sequence specific manner but exploit different mechanisms. Here we describe major developments that have led to the advent of therapeutic oligonucleotides for treatment of TTR-related disease. Full article

(This article belongs to the Special Issue Frontiers in Nucleic Acid Chemistry)

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G-Quadruplex Forming Oligonucleotides as Anti-HIV Agents

by Domenica Musumeci, Claudia Riccardi and Daniela Montesarchio

Molecules 2015, 20(9), 17511-17532; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules200917511 - 22 Sep 2015

Cited by 51 | Viewed by 10000

Abstract

Though a variety of different non-canonical nucleic acids conformations have been recognized, G-quadruplex structures are probably the structural motifs most commonly found within known oligonucleotide-based aptamers. This could be ascribed to several factors, as their large conformational diversity, marked responsiveness of their folding/unfolding [...] Read more.

Though a variety of different non-canonical nucleic acids conformations have been recognized, G-quadruplex structures are probably the structural motifs most commonly found within known oligonucleotide-based aptamers. This could be ascribed to several factors, as their large conformational diversity, marked responsiveness of their folding/unfolding processes to external stimuli, high structural compactness and chemo-enzymatic and thermodynamic stability. A number of G-quadruplex-forming oligonucleotides having relevant in vitro anti-HIV activity have been discovered in the last two decades through either SELEX or rational design approaches. Improved aptamers have been obtained by chemical modifications of natural oligonucleotides, as terminal conjugations with large hydrophobic groups, replacement of phosphodiester linkages with phosphorothioate bonds or other surrogates, insertion of base-modified monomers, etc. In turn, detailed structural studies have elucidated the peculiar architectures adopted by many G-quadruplex-based aptamers and provided insight into their mechanism of action. An overview of the state-of-the-art knowledge of the relevance of putative G-quadruplex forming sequences within the viral genome and of the most studied G-quadruplex-forming aptamers, selectively targeting HIV proteins, is here presented. Full article

(This article belongs to the Special Issue Frontiers in Nucleic Acid Chemistry)

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