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12 pages, 1352 KiB

Open AccessArticle

Yeast-Derived Nucleotides Enhance Fibroblast Migration and Proliferation and Provide Clinical Benefits in Atopic Dermatitis

by Sergi Segarra, Ivica Bošnjak, Igor Mioč, Bojana Čurčija, Vlatka-Antonija Csik, Srećko Krešić, Jessica Romero-Rueda, Anna Rodríguez and Daniel Martínez-Puig

Int. J. Mol. Sci. 2024, 25(5), 2890; https://doi.org/10.3390/ijms25052890 - 01 Mar 2024

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Abstract

Nucleotides, glycosaminoglycans, and omega-3 essential fatty acids (O3s) could be used for improving skin health, although their modes of action, alone or in combination, are not yet fully understood. To gain some insight into these mechanisms, we performed two in vitro tests and [...] Read more.

Nucleotides, glycosaminoglycans, and omega-3 essential fatty acids (O3s) could be used for improving skin health, although their modes of action, alone or in combination, are not yet fully understood. To gain some insight into these mechanisms, we performed two in vitro tests and one in vivo pilot trial. The effects on human dermal fibroblast proliferation and migration were evaluated with the following compounds and combinations: 0.156 mg/mL O3s, 0.0017 mg/mL hyaluronic acid (HA), 0.0004 mg/mL dermatan sulfate (DS), 0.0818 mg/mL nucleotides, and [O3s + HA + DS] and [O3s + HA + DS + nucleotides] at the same concentrations. In both in vitro assays, adding nucleotides to [O3s + HA + DS] provided significant improvements. The resulting combination [O3s + HA + DS + nucleotides] was then tested in vivo in dogs with atopic dermatitis by oral administration of a supplement providing a daily amount of 40 mg/kg nucleotides, 0.9 mg/kg HA, 0.18 mg/kg DS, 53.4 mg/kg EPA, and 7.6 mg/kg DHA. After 30 days, the pruritus visual analog scale (pVAS) score was significantly reduced, and no adverse effects were observed. In conclusion, the combination of nucleotides plus glycosaminoglycans and O3s could serve as a useful therapeutic alternative in skin health applications. Full article

(This article belongs to the Special Issue Advanced Research on Nucleic Acids: Therapeutic Potential and Applications)

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17 pages, 2741 KiB

Open AccessArticle

Effective Antiviral Application of Antisense in Plants by Exploiting Accessible Sites in the Target RNA

by Cornelia Gruber, Torsten Gursinsky, Selma Gago-Zachert, Vitantonio Pantaleo and Sven-Erik Behrens

Int. J. Mol. Sci. 2023, 24(24), 17153; https://doi.org/10.3390/ijms242417153 - 05 Dec 2023

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Abstract

Antisense oligodeoxynucleotides (ASOs) have long been used to selectively inhibit or modulate gene expression at the RNA level, and some ASOs are approved for clinical use. However, the practicability of antisense technologies remains limited by the difficulty of reliably predicting the sites accessible [...] Read more.

Antisense oligodeoxynucleotides (ASOs) have long been used to selectively inhibit or modulate gene expression at the RNA level, and some ASOs are approved for clinical use. However, the practicability of antisense technologies remains limited by the difficulty of reliably predicting the sites accessible to ASOs in complex folded RNAs. Recently, we applied a plant-based method that reproduces RNA-induced RNA silencing in vitro to reliably identify sites in target RNAs that are accessible to small interfering RNA (siRNA)-guided Argonaute endonucleases. Here, we show that this method is also suitable for identifying ASOs that are effective in DNA-induced RNA silencing by RNases H. We show that ASOs identified in this way that target a viral genome are comparably effective in protecting plants from infection as siRNAs with the corresponding sequence. The antiviral activity of the ASOs could be further enhanced by chemical modification. This led to two important conclusions: siRNAs and ASOs that can effectively knock down complex RNA molecules can be identified using the same approach, and ASOs optimized in this way could find application in crop protection. The technology developed here could be useful not only for effective RNA silencing in plants but also in other organisms. Full article

(This article belongs to the Special Issue Advanced Research on Nucleic Acids: Therapeutic Potential and Applications)

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14 pages, 2656 KiB

Open AccessArticle

Improving the Biological Properties of Thrombin-Binding Aptamer by Incorporation of 8-Bromo-2′-Deoxyguanosine and 2′-Substituted RNA Analogues

by Antonella Virgilio, Daniela Benigno, Carla Aliberti, Valentina Vellecco, Mariarosaria Bucci, Veronica Esposito and Aldo Galeone

Int. J. Mol. Sci. 2023, 24(21), 15529; https://doi.org/10.3390/ijms242115529 - 24 Oct 2023

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Abstract

Thrombin-binding aptamer (TBA) is one of the best-known G-quadruplex (G4)-forming aptamers. By adopting its peculiar chair-like G4 structure, TBA can efficiently bind to thrombin, thus producing an anticoagulant effect. The major limit to its therapeutic application is represented by its poor thermal and [...] Read more.

Thrombin-binding aptamer (TBA) is one of the best-known G-quadruplex (G4)-forming aptamers. By adopting its peculiar chair-like G4 structure, TBA can efficiently bind to thrombin, thus producing an anticoagulant effect. The major limit to its therapeutic application is represented by its poor thermal and biological resistance. Therefore, numerous research studies have been focused on the design of TBA analogues with chemical modifications to improve its pharmacokinetic and pharmacodynamic properties. To maintain the functional recognition to protein surface on which TBA anticoagulant activity depends, it is essential to preserve the canonical antiparallel topology of the TBA quadruplex core. In this paper, we have designed three TBA variants with modified G-tetrads to evaluate the effects of nucleobase and sugar moiety chemical modifications on biological properties of TBA, preserving its chair-like G-quadruplex structure. All derivatives contain 8-bromo-2′-deoxyguanosine (G^Br) in syn positions, while in the anti-positions, locked nucleic acid guanosine (G^LNA) in the analogue TBABL, 2’-O-methylguanosine (G^OMe) in TBABM, and 2’-F-riboguanosine (G^F) in TBABF is present. CD (Circular Dichroism), CD melting, 1H-NMR (Nuclear Magnetic Resonance), and non-denaturing PAGE (Polyacrylamide Gel Electrophoresis), nuclease stability, prothrombin time (PT) and fibrinogen-clotting assays have been performed to investigate the structural and biological properties of these TBA analogues. The most interesting results have been obtained with TBABF, which revealed extraordinary thermal stability (T_m approximately 40 °C higher than that of TBA), anticoagulant activity almost doubled compared to the original aptamer, and, above all, a never-observed resistance to nucleases, as 50% of its G4 species was still present in 50% FBS at 24 h. These data indicate TBABF as one of the best TBA analogue ever designed and investigated, to the best of our knowledge, overcoming the main limitations to therapeutic applications of this aptamer. Full article

(This article belongs to the Special Issue Advanced Research on Nucleic Acids: Therapeutic Potential and Applications)

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11 pages, 1506 KiB

Open AccessArticle

Quantification of Oligonucleotides Using Tandem Mass Spectrometry with Isobaric Internal Standards

by Christopher Gawlig, Güngör Hanci and Michael Rühl

Int. J. Mol. Sci. 2023, 24(19), 14691; https://doi.org/10.3390/ijms241914691 - 28 Sep 2023

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Abstract

In recent years, oligonucleotides have become more important in research, drug approvals and medical therapies. Due to this growing interest in pharmaceutical applications, it is essential to develop reliable analytical methods for this substance class. In this work, we present a quantification method [...] Read more.

In recent years, oligonucleotides have become more important in research, drug approvals and medical therapies. Due to this growing interest in pharmaceutical applications, it is essential to develop reliable analytical methods for this substance class. In this work, we present a quantification method using liquid chromatography coupled with tandem mass spectrometry by applying an isobaric oligonucleotide standard. In addition to a proof of principle, we perform a method qualification to assess its readiness for validation according to ICH Q2 guidelines. In addition to good linearity, sensitivity, accuracy and recovery, the method showed no significant matrix effects. Furthermore, we demonstrated the application of the method by applying the quantification in a biological matrix, as well as an exemplary degradation of an oligonucleotide in bovine plasma. Full article

(This article belongs to the Special Issue Advanced Research on Nucleic Acids: Therapeutic Potential and Applications)

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17 pages, 4442 KiB

Open AccessArticle

Myogenetic Oligodeoxynucleotide Induces Myocardial Differentiation of Murine Pluripotent Stem Cells

by Mina Ishioka, Yuma Nihashi, Yoichi Sunagawa, Koji Umezawa, Takeshi Shimosato, Hiroshi Kagami, Tatsuya Morimoto and Tomohide Takaya

Int. J. Mol. Sci. 2023, 24(18), 14380; https://doi.org/10.3390/ijms241814380 - 21 Sep 2023

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Abstract

An 18-base myogenetic oligodeoxynucleotide (myoDN), iSN04, acts as an anti-nucleolin aptamer and induces myogenic differentiation of skeletal muscle myoblasts. This study investigated the effect of iSN04 on murine embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). In the undifferentiated state, iSN04 [...] Read more.

An 18-base myogenetic oligodeoxynucleotide (myoDN), iSN04, acts as an anti-nucleolin aptamer and induces myogenic differentiation of skeletal muscle myoblasts. This study investigated the effect of iSN04 on murine embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). In the undifferentiated state, iSN04 inhibited the proliferation of ESCs and iPSCs but did not affect the expression of pluripotent markers. In the differentiating condition, iSN04 treatment of ESCs/iPSCs from day 5 onward dramatically induced differentiation into Nkx2-5⁺ beating cardiomyocytes with upregulation of Gata4, Isl1, and Nkx2-5, whereas iSN04 treatment from earlier stages completely inhibited cardiomyogenesis. RNA sequencing revealed that iSN04 treatment from day 5 onward contributes to the generation of cardiac progenitors by modulating the Wnt signaling pathway. Immunostaining showed that iSN04 suppressed the cytoplasmic translocation of nucleolin and restricted it to the nucleoli. These results demonstrate that nucleolin inhibition by iSN04 facilitates the terminal differentiation of cardiac mesoderm into cardiomyocytes but interferes with the differentiation of early mesoderm into the cardiac lineage. This is the first report on the generation of cardiomyocytes from pluripotent stem cells using a DNA aptamer. Since iSN04 did not induce hypertrophic responses in primary-cultured cardiomyocytes, iSN04 would be useful and safe for the regenerative therapy of heart failure using stem cell-derived cardiomyocytes. Full article

(This article belongs to the Special Issue Advanced Research on Nucleic Acids: Therapeutic Potential and Applications)

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21 pages, 7155 KiB

Open AccessArticle

Exploring the Interaction of New Pyridoquinazoline Derivatives with G-Quadruplex in the c-MYC Promoter Region

by Salvatore Princiotto, Maria Karelou, Rachel Ioannidi, Giovanni Luca Beretta, Nadia Zaffaroni, Roberto Artali, Ioannis K. Kostakis, Stefania Mazzini and Sabrina Dallavalle

Int. J. Mol. Sci. 2023, 24(18), 14346; https://doi.org/10.3390/ijms241814346 - 20 Sep 2023

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Abstract

Novel amino-substituted pyridoquinazolinone derivatives have been designed and synthesized as potential c-MYC G-quadruplex (G4) ligands, employing an efficient methodology. All the new compounds exhibited moderate to good antiproliferative activity against the human osteosarcoma U2OS cell line. NMR and docking experiments revealed that the [...] Read more.

Novel amino-substituted pyridoquinazolinone derivatives have been designed and synthesized as potential c-MYC G-quadruplex (G4) ligands, employing an efficient methodology. All the new compounds exhibited moderate to good antiproliferative activity against the human osteosarcoma U2OS cell line. NMR and docking experiments revealed that the recently synthesized compounds interact with the Pu22 G-quadruplex in the c-MYC promoter region, establishing a 2:1 complex, with each molecule positioned over the tetrads at the 3′- and 5′-ends. Full article

(This article belongs to the Special Issue Advanced Research on Nucleic Acids: Therapeutic Potential and Applications)

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14 pages, 3695 KiB

Open AccessArticle

A Unique G-Quadruplex Aptamer: A Novel Approach for Cancer Cell Recognition, Cell Membrane Visualization, and RSV Infection Detection

by Chao-Da Xiao, Ming-Qing Zhong, Yue Gao, Zheng-Lin Yang, Meng-Hao Jia, Xiao-Hui Hu, Yan Xu and Xiang-Chun Shen

Int. J. Mol. Sci. 2023, 24(18), 14344; https://doi.org/10.3390/ijms241814344 - 20 Sep 2023

Cited by 1 | Viewed by 1113

Abstract

Surface staining has emerged as a rapid technique for applying external stains to trace cellular identities in diverse populations. In this study, we developed a distinctive aptamer with selective binding to cell surface nucleolin (NCL), bypassing cytoplasmic internalization. Conjugation of the aptamer with [...] Read more.

Surface staining has emerged as a rapid technique for applying external stains to trace cellular identities in diverse populations. In this study, we developed a distinctive aptamer with selective binding to cell surface nucleolin (NCL), bypassing cytoplasmic internalization. Conjugation of the aptamer with a FAM group facilitated NCL visualization on live cell surfaces with laser confocal microscopy. To validate the aptamer-NCL interaction, we employed various methods, including the surface plasmon resonance, IHC-based flow cytometry, and electrophoretic mobility shift assay. The G-quadruplex formations created by aptamers were confirmed with a nuclear magnetic resonance and an electrophoretic mobility shift assay utilizing BG4, a G-quadruplex-specific antibody. Furthermore, the aptamer exhibited discriminatory potential in distinguishing between cancerous and normal cells using flow cytometry. Notably, it functioned as a dynamic probe, allowing real-time monitoring of heightened NCL expression triggered by a respiratory syncytial virus (RSV) on normal cell surfaces. This effect was subsequently counteracted with dsRNA transfection and suppressed the NCL expression; thus, emphasizing the dynamic attributes of the probe. These collective findings highlight the robust versatility of our aptamer as a powerful tool for imaging cell surfaces, holding promising implications for cancer cell identification and the detection of RSV infections. Full article

(This article belongs to the Special Issue Advanced Research on Nucleic Acids: Therapeutic Potential and Applications)

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Review

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32 pages, 2995 KiB

Open AccessReview

Subverting the Canon: Novel Cancer-Promoting Functions and Mechanisms for snoRNAs

by Matthew Huo, Sudhir Kumar Rai, Ken Nakatsu, Youping Deng and Mayumi Jijiwa

Int. J. Mol. Sci. 2024, 25(5), 2923; https://doi.org/10.3390/ijms25052923 - 02 Mar 2024

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Abstract

Small nucleolar RNAs (snoRNAs) constitute a class of intron-derived non-coding RNAs ranging from 60 to 300 nucleotides. Canonically localized in the nucleolus, snoRNAs play a pivotal role in RNA modifications and pre-ribosomal RNA processing. Based on the types of modifications they involve, such [...] Read more.

Small nucleolar RNAs (snoRNAs) constitute a class of intron-derived non-coding RNAs ranging from 60 to 300 nucleotides. Canonically localized in the nucleolus, snoRNAs play a pivotal role in RNA modifications and pre-ribosomal RNA processing. Based on the types of modifications they involve, such as methylation and pseudouridylation, they are classified into two main families—box C/D and H/ACA snoRNAs. Recent investigations have revealed the unconventional synthesis and biogenesis strategies of snoRNAs, indicating their more profound roles in pathogenesis than previously envisioned. This review consolidates recent discoveries surrounding snoRNAs and provides insights into their mechanistic roles in cancer. It explores the intricate interactions of snoRNAs within signaling pathways and speculates on potential therapeutic solutions emerging from snoRNA research. In addition, it presents recent findings on the long non-coding small nucleolar RNA host gene (lncSNHG), a subset of long non-coding RNAs (lncRNAs), which are the transcripts of parental SNHGs that generate snoRNA. The nucleolus, the functional epicenter of snoRNAs, is also discussed. Through a deconstruction of the pathways driving snoRNA-induced oncogenesis, this review aims to serve as a roadmap to guide future research in the nuanced field of snoRNA–cancer interactions and inspire potential snoRNA-related cancer therapies. Full article

(This article belongs to the Special Issue Advanced Research on Nucleic Acids: Therapeutic Potential and Applications)

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