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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://doi.org/10.3390/molecules27206831 - 12 Oct 2022

Viewed by 1705

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://doi.org/10.3390/molecules23092162 - 28 Aug 2018

Cited by 16 | Viewed by 4630

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://doi.org/10.3390/molecules23081874 - 27 Jul 2018

Cited by 11 | Viewed by 5487

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://doi.org/10.3390/molecules23071806 - 21 Jul 2018

Cited by 7 | Viewed by 4651

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://doi.org/10.3390/molecules22111831 - 27 Oct 2017

Cited by 13 | Viewed by 5517

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://doi.org/10.3390/molecules22060993 - 15 Jun 2017

Cited by 5 | Viewed by 4853

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://doi.org/10.3390/molecules21091212 - 10 Sep 2016

Cited by 17 | Viewed by 7504

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://doi.org/10.3390/molecules21091082 - 23 Aug 2016

Cited by 5 | Viewed by 7835

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://doi.org/10.3390/molecules21060766 - 11 Jun 2016

Cited by 3 | Viewed by 7584

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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2165 KiB

Open AccessArticle

Identification of RNA Oligonucleotides Binding to Several Proteins from Potential G-Quadruplex Forming Regions in Transcribed Pre-mRNA

by Taiki Saito, Wataru Yoshida, Tomomi Yokoyama, Koichi Abe and Kazunori Ikebukuro

Molecules 2015, 20(11), 20832-20840; https://doi.org/10.3390/molecules201119733 - 23 Nov 2015

Cited by 7 | Viewed by 6765

Abstract

G-quadruplexes (G4s) are noncanonical DNA/RNA structures formed by guanine-rich sequences. Recently, G4s have been found not only in aptamers but also in the genomic DNA and transcribed RNA. In this study, we identified new RNA oligonucleotides working as aptamers by focusing on G4-forming [...] Read more.

G-quadruplexes (G4s) are noncanonical DNA/RNA structures formed by guanine-rich sequences. Recently, G4s have been found not only in aptamers but also in the genomic DNA and transcribed RNA. In this study, we identified new RNA oligonucleotides working as aptamers by focusing on G4-forming RNAs located within the pre-mRNA. We showed that the G4 in the 5′ UTR and first intron of VEGFA bound to the protein encoded in VEGFA gene, VEGF165, with high affinity. Moreover, G4-forming RNAs located within the PDGFA and the PDGFB introns bound to PDGF-AA and PDGF-BB, respectively, indicating that G4 in the pre-mRNA could be an aptamer. It had been reported that the putative G4-forming RNA sequences are located in some parts of most genes, thus our strategy for aptamer identification could be applicable to other proteins. It has been reported that some G4-forming RNAs in 5′ UTRs are involved in translation control; however, G4-forming excised intronic RNA function has not been revealed previously. Therefore, these findings could not only contribute to the identification of RNA aptamers but also provide new insights into the biological functioning of G4-forming RNAs located within intronic RNA sequences. Full article

(This article belongs to the Special Issue Aptamers: Past, Present, and Future)

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932 KiB

Open AccessArticle

Fluorescent Sensor for PH Monitoring Based on an i-Motif- – Switching Aptamer Containing a Tricyclic Cytosine Analogue (tC)

by Patrycja Bielecka and Bernard Juskowiak

Molecules 2015, 20(10), 18511-18525; https://doi.org/10.3390/molecules201018511 - 9 Oct 2015

Cited by 22 | Viewed by 7802

Abstract

There are cytosine-rich regions in the genome that bind protons with high specificity. Thus protonated C-rich sequence may undergo folding to tetraplex structures called i-motifs. Therefore, one can regard such specific C-rich oligonucleotides as aptamers that recognize protons and undergo conformational transitions. Proper [...] Read more.

There are cytosine-rich regions in the genome that bind protons with high specificity. Thus protonated C-rich sequence may undergo folding to tetraplex structures called i-motifs. Therefore, one can regard such specific C-rich oligonucleotides as aptamers that recognize protons and undergo conformational transitions. Proper labeling of the aptamer with a fluorescent tag constitutes a platform to construct a pH-sensitive aptasensor. Since the hemiprotonated C-C⁺ base pairs are responsible for the folded tetraplex structure of i-motif, we decided to substitute one of cytosines in an aptamer sequence with its fluorescent analogue, 1,3-diaza-2-oxophenothiazine (tC). In this paper we report on three tC-modified fluorescent probes that contain RET related sequences as a proton recognizing aptamer. Results of the circular dichroism (CD), UV absorption melting experiments, and steady-state fluorescence measurements of these tC-modified i-motif probes are presented and discussed. The pH-induced i-motif formation by the probes resulted in fluorescence quenching of tC fluorophore. Efficiency of quenching was related to the pH variations. Suitability of the sensor for monitoring pH changes was also demonstrated. Full article

(This article belongs to the Special Issue Aptamers: Past, Present, and Future)

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1043 KiB

Open AccessArticle

RNA Aptamers as Molecular Tools to Study the Functionality of the Hepatitis C Virus CRE Region

by Alba Fernández-Sanlés, Beatriz Berzal-Herranz, Rodrigo González-Matamala, Pablo Ríos-Marco, Cristina Romero-López and Alfredo Berzal-Herranz

Molecules 2015, 20(9), 16030-16047; https://doi.org/10.3390/molecules200916030 - 2 Sep 2015

Cited by 9 | Viewed by 6251

Abstract

Background: Hepatitis C virus (HCV) contains a (+) ssRNA genome with highly conserved structural, functional RNA domains, many of them with unknown roles for the consecution of the viral cycle. Such genomic domains are candidate therapeutic targets. This study reports the functional characterization [...] Read more.

Background: Hepatitis C virus (HCV) contains a (+) ssRNA genome with highly conserved structural, functional RNA domains, many of them with unknown roles for the consecution of the viral cycle. Such genomic domains are candidate therapeutic targets. This study reports the functional characterization of a set of aptamers targeting the cis-acting replication element (CRE) of the HCV genome, an essential partner for viral replication and also involved in the regulation of protein synthesis. Methods: Forty-four aptamers were tested for their ability to interfere with viral RNA synthesis in a subgenomic replicon system. Some of the most efficient inhibitors were further evaluated for their potential to affect the recruitment of the HCV RNA-dependent RNA polymerase (NS5B) and the viral translation in cell culture. Results: Four aptamers emerged as potent inhibitors of HCV replication by direct interaction with functional RNA domains of the CRE, yielding a decrease in the HCV RNA levels higher than 90%. Concomitantly, one of them also induced a significant increase in viral translation (>50%). The three remaining aptamers efficiently competed with the binding of the NS5B protein to the CRE. Conclusions: Present findings confirm the potential of the CRE as an anti-HCV target and support the use of aptamers as molecular tools for investigating the functionality of RNA domains in viral genomes. Full article

(This article belongs to the Special Issue Aptamers: Past, Present, and Future)

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Review

Jump to: Research

18 pages, 1283 KiB

Open AccessReview

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://doi.org/10.3390/molecules28041517 - 4 Feb 2023

Cited by 4 | Viewed by 2960

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://doi.org/10.3390/molecules23010124 - 8 Jan 2018

Cited by 25 | Viewed by 8608

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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31930 KiB

Open AccessFeature PaperReview

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://doi.org/10.3390/molecules22122108 - 30 Nov 2017

Cited by 38 | Viewed by 10490

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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Open AccessReview

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

by Prabodhika Mallikaratchy

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

Cited by 75 | Viewed by 10128

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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2132 KiB

Open AccessReview

Methods for Improving Aptamer Binding Affinity

by Hijiri Hasegawa, Nasa Savory, Koichi Abe and Kazunori Ikebukuro

Molecules 2016, 21(4), 421; https://doi.org/10.3390/molecules21040421 - 28 Mar 2016

Cited by 187 | Viewed by 14822

Abstract

Aptamers are single stranded oligonucleotides that bind a wide range of biological targets. Although aptamers can be isolated from pools of random sequence oligonucleotides using affinity-based selection, aptamers with high affinities are not always obtained. Therefore, further refinement of aptamers is required to [...] Read more.

Aptamers are single stranded oligonucleotides that bind a wide range of biological targets. Although aptamers can be isolated from pools of random sequence oligonucleotides using affinity-based selection, aptamers with high affinities are not always obtained. Therefore, further refinement of aptamers is required to achieve desired binding affinities. The optimization of primary sequences and stabilization of aptamer conformations are the main approaches to refining the binding properties of aptamers. In particular, sequence optimization using combined in silico sequence recombinations and in vitro functional evaluations is effective for the improvement of binding affinities, however, the binding affinities of aptamers are limited by the low hydrophobicity of nucleic acids. Accordingly, introduction of hydrophobic moieties into aptamers expands the diversity of interactions between aptamers and targets. Moreover, construction of multivalent aptamers by connecting aptamers that recognize distinct epitopes is an attractive approach to substantial increases in binding affinity. In addition, binding affinities can be tuned by optimizing the scaffolds of multivalent constructs. In this review, we summarize the various techniques for improving the binding affinities of aptamers. Full article

(This article belongs to the Special Issue Aptamers: Past, Present, and Future)

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Open AccessReview

Oligonucleotide Functionalised Microbeads: Indispensable Tools for High-Throughput Aptamer Selection

by Lewis A. Fraser, Andrew B. Kinghorn, Marco S. L. Tang, Yee-Wai Cheung, Bryce Lim, Shaolin Liang, Roderick M. Dirkzwager and Julian A. Tanner

Molecules 2015, 20(12), 21298-21312; https://doi.org/10.3390/molecules201219766 - 1 Dec 2015

Cited by 15 | Viewed by 8698

Abstract

The functionalisation of microbeads with oligonucleotides has become an indispensable technique for high-throughput aptamer selection in SELEX protocols. In addition to simplifying the separation of binding and non-binding aptamer candidates, microbeads have facilitated the integration of other technologies such as emulsion PCR (ePCR) [...] Read more.

The functionalisation of microbeads with oligonucleotides has become an indispensable technique for high-throughput aptamer selection in SELEX protocols. In addition to simplifying the separation of binding and non-binding aptamer candidates, microbeads have facilitated the integration of other technologies such as emulsion PCR (ePCR) and Fluorescence Activated Cell Sorting (FACS) to high-throughput selection techniques. Within these systems, monoclonal aptamer microbeads can be individually generated and assayed to assess aptamer candidate fitness thereby helping eliminate stochastic effects which are common to classical SELEX techniques. Such techniques have given rise to aptamers with 1000 times greater binding affinities when compared to traditional SELEX. Another emerging technique is Fluorescence Activated Droplet Sorting (FADS) whereby selection does not rely on binding capture allowing evolution of a greater diversity of aptamer properties such as fluorescence or enzymatic activity. Within this review we explore examples and applications of oligonucleotide functionalised microbeads in aptamer selection and reflect upon new opportunities arising for aptamer science. Full article

(This article belongs to the Special Issue Aptamers: Past, Present, and Future)

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Open AccessReview

DNA Aptamers in the Diagnosis and Treatment of Human Diseases

by Qinchang Zhu, Ge Liu and Masaaki Kai

Molecules 2015, 20(12), 20979-20997; https://doi.org/10.3390/molecules201219739 - 25 Nov 2015

Cited by 101 | Viewed by 11717

Abstract

Aptamers have a promising role in the field of life science and have been extensively researched for application as analytical tools, therapeutic agents and as vehicles for targeted drug delivery. Compared with RNA aptamers, DNA aptamers have inherent advantages in stability and facility [...] Read more.

Aptamers have a promising role in the field of life science and have been extensively researched for application as analytical tools, therapeutic agents and as vehicles for targeted drug delivery. Compared with RNA aptamers, DNA aptamers have inherent advantages in stability and facility of generation and synthesis. To better understand the specific potential of DNA aptamers, an overview of the progress in the generation and application of DNA aptamers in human disease diagnosis and therapy are presented in this review. Special attention is given to researches that are relatively close to practical application. DNA aptamers are expected to have great potential in the diagnosis and treatment of human diseases. Full article

(This article belongs to the Special Issue Aptamers: Past, Present, and Future)

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Open AccessReview

Generation of Aptamers with an Expanded Chemical Repertoire

by Stella Diafa and Marcel Hollenstein

Molecules 2015, 20(9), 16643-16671; https://doi.org/10.3390/molecules200916643 - 14 Sep 2015

Cited by 91 | Viewed by 10568

Abstract

The enzymatic co-polymerization of modified nucleoside triphosphates (dN*TPs and N*TPs) is a versatile method for the expansion and exploration of expanded chemical space in SELEX and related combinatorial methods of in vitro selection. This strategy can be exploited to generate aptamers with improved [...] Read more.

The enzymatic co-polymerization of modified nucleoside triphosphates (dN*TPs and N*TPs) is a versatile method for the expansion and exploration of expanded chemical space in SELEX and related combinatorial methods of in vitro selection. This strategy can be exploited to generate aptamers with improved or hitherto unknown properties. In this review, we discuss the nature of the functionalities appended to nucleoside triphosphates and their impact on selection experiments. The properties of the resulting modified aptamers will be described, particularly those integrated in the fields of biomolecular diagnostics, therapeutics, and in the expansion of genetic systems (XNAs). Full article

(This article belongs to the Special Issue Aptamers: Past, Present, and Future)

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Open AccessReview

A Highlight of Recent Advances in Aptamer Technology and Its Application

by Hongguang Sun and Youli Zu

Molecules 2015, 20(7), 11959-11980; https://doi.org/10.3390/molecules200711959 - 30 Jun 2015

Cited by 257 | Viewed by 24903

Abstract

Aptamers and SELEX (systematic evolution of ligands by exponential enrichment) technology have gained increasing attention over the past 25 years. Despite their functional similarity to protein antibodies, oligonucleotide aptamers have many unique properties that are suitable for clinical applications and industrialization. Aptamers may [...] Read more.

Aptamers and SELEX (systematic evolution of ligands by exponential enrichment) technology have gained increasing attention over the past 25 years. Despite their functional similarity to protein antibodies, oligonucleotide aptamers have many unique properties that are suitable for clinical applications and industrialization. Aptamers may be superior to antibodies in fields such as biomarker discovery, in vitro and in vivo diagnosis, precisely controlled drug release, and targeted therapy. However, aptamer commercialization has not occurred as quickly as expected, and few aptamer-based products have yet successfully entered clinical and industrial use. Thus, it is important to critically review some technical barriers of aptamer and SELEX technology per se that may impede aptamer development and application. To date, how to rapidly obtain aptamers with superior bioavailability over antibodies remains the key issue. In this review, we discuss different chemical and structural modification strategies aimed to enhance aptamer bioavailability. We also discuss improvements to SELEX process steps to shorten the selection period and improve the SELEX process success rate. Applications in which aptamers are particularly suited and perform differently or superior to antibodies are briefly introduced. Full article

(This article belongs to the Special Issue Aptamers: Past, Present, and Future)

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Predicting the Uncertain Future of Aptamer-Based Diagnostics and Therapeutics

by John G. Bruno

Molecules 2015, 20(4), 6866-6887; https://doi.org/10.3390/molecules20046866 - 16 Apr 2015

Cited by 118 | Viewed by 12925

Abstract

Despite the great promise of nucleic acid aptamers in the areas of diagnostics and therapeutics for their facile in vitro development, lack of immunogenicity and other desirable properties, few truly successful aptamer-based products exist in the clinical or other markets. Core reasons for [...] Read more.

Despite the great promise of nucleic acid aptamers in the areas of diagnostics and therapeutics for their facile in vitro development, lack of immunogenicity and other desirable properties, few truly successful aptamer-based products exist in the clinical or other markets. Core reasons for these commercial deficiencies probably stem from industrial commitment to antibodies including a huge financial investment in humanized monoclonal antibodies and a general ignorance about aptamers and their performance among the research and development community. Given the early failures of some strong commercial efforts to gain government approval and bring aptamer-based products to market, it may seem that aptamers are doomed to take a backseat to antibodies forever. However, the key advantages of aptamers over antibodies coupled with niche market needs that only aptamers can fill and more recent published data still point to a bright commercial future for aptamers in areas such as infectious disease and cancer diagnostics and therapeutics. As more researchers and entrepreneurs become familiar with aptamers, it seems inevitable that aptamers will at least be considered for expanded roles in diagnostics and therapeutics. This review also examines new aptamer modifications and attempts to predict new aptamer applications that could revolutionize biomedical technology in the future and lead to marketed products. Full article

(This article belongs to the Special Issue Aptamers: Past, Present, and Future)

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