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Aptamers: Functional-Structural Studies and Biomedical Applications 2.0

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A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Biophysics".

Deadline for manuscript submissions: closed (30 August 2023) | Viewed by 29346

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Prof. Dr. Filomena Sica

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Department of Chemical Sciences, University of Naples Federico II, 80126 Naples, Italy
Interests: X-ray biocrystallography; structure–function relationship; protein structures; G-quadruplex oligonucleotides; protein–aptamer complexes; spectroscopic methods; molecular dynamics simulations
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Dr. Romualdo Troisi

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Dear Colleagues,

Macromolecular recognition is the origin of biological specificity. It takes place in a crowded noisy environment and requires molecular selectivity and affinity. In this context, a special class of synthetic ligands is represented by aptamers, which are oligonucleotides or peptides that bind almost any molecule of choice with high affinity and selectivity. In particular, aptamers are able to modulate the function of most proteins, including blood-clotting factors, cell-surface receptors, and transcription factors. These ligands are characterized by low immunogenicity, a well-established synthesis protocol, and chemical modification technology, which allow a fine-tuning of their bioavailability and pharmacokinetics, thus extending their therapeutic potential. These same features make aptamers excellent candidates also in the development of effective biomedical sensors. Despite numerous studies on aptamers and their interaction with the target molecules, there is still much work to do in this field to improve the pharmacological and diagnostic properties of these ligands.

This Special Issue will concern a selection of original research, review articles, and commentaries focused on functional-structural studies and biomedical applications of aptamers, in a joint attempt to highlight the merits and to overcome the current limitations of this technology.

Prof. Dr. Filomena Sica
Dr. Romualdo Troisi
Guest Editors

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Keywords

aptamer therapeutics
oligonucleotide aptamer
peptide aptamer
biological modulation
aptamer-target complexes
modified oligonucleotides
structure and dynamics
protein activity modulation
aptamer-based detection
biosensor
biomedical application
clinical diagnostics

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Published Papers (12 papers)

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Editorial

Jump to: Research, Review

3 pages, 142 KiB

Open AccessEditorial

Aptamers: Functional-Structural Studies and Biomedical Applications 2.0

by Romualdo Troisi and Filomena Sica

Int. J. Mol. Sci. 2024, 25(8), 4279; https://doi.org/10.3390/ijms25084279 - 12 Apr 2024

Viewed by 770

Abstract

As a follow-up to the previous Special Issue “Aptamers: Functional-Structural Studies and Biomedical Applications” [...] Full article

(This article belongs to the Special Issue Aptamers: Functional-Structural Studies and Biomedical Applications 2.0)

Research

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11 pages, 2460 KiB

Open AccessArticle

TBA for Sensing Toxic Cations: A Critical Analysis of Structural and Electrical Properties

by Eleonora Alfinito

Int. J. Mol. Sci. 2023, 24(19), 14492; https://doi.org/10.3390/ijms241914492 - 23 Sep 2023

Cited by 1 | Viewed by 1257

Abstract

Food and drinks can be contaminated with pollutants such as lead and strontium, which poses a serious danger to human health. For this reason, a number of effective sensors have been developed for the rapid and highly selective detection of such contaminants. TBA, a well-known aptamer developed to selectively target and thereby inhibit the protein of clinical interest α-thrombin, is receiving increasing attention for sensing applications, particularly for the sensing of different cations. Indeed, TBA, in the presence of these cations, folds into the stable G-quadruplex structure. Furthermore, different cations produce small but significant changes in this structure that result in changes in the electrical responses that TBA can produce. In this article, we give an overview of the expected data regarding the use of TBA in the detection of lead and strontium, calculating the expected electrical response using different measurement techniques. Finally, we conclude that TBA should be able to detect strontium with a sensitivity approximately double that achievable for lead. Full article

(This article belongs to the Special Issue Aptamers: Functional-Structural Studies and Biomedical Applications 2.0)

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15 pages, 2170 KiB

Open AccessArticle

Structural Optimization and Interaction Study of a DNA Aptamer to L1 Cell Adhesion Molecule

by Zhenhao Long, Tao Bing, Xiangru Zhang, Jing Sheng, Shuang Zu, Weiwei Li, Xiangjun Liu, Nan Zhang and Dihua Shangguan

Int. J. Mol. Sci. 2023, 24(10), 8612; https://doi.org/10.3390/ijms24108612 - 11 May 2023

Cited by 5 | Viewed by 2496

Abstract

The L1 cell adhesion molecule (L1CAM) plays important roles in the development and plasticity of the nervous system as well as in tumor formation, progression, and metastasis. New ligands are necessary tools for biomedical research and the detection of L1CAM. Here, DNA aptamer yly12 against L1CAM was optimized to have much stronger binding affinity (10–24 fold) at room temperature and 37 °C via sequence mutation and extension. This interaction study revealed that the optimized aptamers (yly20 and yly21) adopted a hairpin structure containing two loops and two stems. The key nucleotides for aptamer binding mainly located in loop I and its adjacent area. Stem I mainly played the role of stabilizing the binding structure. The yly-series aptamers were demonstrated to bind the Ig6 domain of L1CAM. This study reveals a detailed molecular mechanism for the interaction between yly-series aptamers and L1CAM and provides guidance for drug development and detection probe design against L1CAM. Full article

(This article belongs to the Special Issue Aptamers: Functional-Structural Studies and Biomedical Applications 2.0)

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19 pages, 2722 KiB

Open AccessArticle

Light-Up Split Broccoli Aptamer as a Versatile Tool for RNA Assembly Monitoring in Cell-Free TX-TL Systems, Hybrid RNA/DNA Origami Tagging and DNA Biosensing

by Emanuela Torelli, Ben Shirt-Ediss, Silvia A. Navarro, Marisa Manzano, Priya Vizzini and Natalio Krasnogor

Int. J. Mol. Sci. 2023, 24(10), 8483; https://doi.org/10.3390/ijms24108483 - 9 May 2023

Cited by 5 | Viewed by 3497

Abstract

Binary light-up aptamers are intriguing and emerging tools with potential in different fields. Herein, we demonstrate the versatility of a split Broccoli aptamer system able to turn on the fluorescence signal only in the presence of a complementary sequence. First, an RNA three-way junction harbouring the split system is assembled in an E. coli-based cell-free TX-TL system where the folding of the functional aptamer is demonstrated. Then, the same strategy is introduced into a ‘bio-orthogonal’ hybrid RNA/DNA rectangle origami characterized by atomic force microscopy: the activation of the split system through the origami self-assembly is demonstrated. Finally, our system is successfully used to detect the femtomoles of a Campylobacter spp. DNA target sequence. Potential applications of our system include the real-time monitoring of the self-assembly of nucleic-acid-based devices in vivo and of the intracellular delivery of therapeutic nanostructures, as well as the in vitro and in vivo detection of different DNA/RNA targets. Full article

(This article belongs to the Special Issue Aptamers: Functional-Structural Studies and Biomedical Applications 2.0)

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13 pages, 3938 KiB

Open AccessArticle

Alpha-Deoxyguanosine to Reshape the Alpha-Thrombin Binding Aptamer

by Natalia A. Kolganova, Vladimir B. Tsvetkov, Andrey A. Stomakhin, Sergei A. Surzhikov, Edward N. Timofeev and Irina V. Varizhuk

Int. J. Mol. Sci. 2023, 24(9), 8406; https://doi.org/10.3390/ijms24098406 - 7 May 2023

Cited by 1 | Viewed by 1738

Abstract

Modification of DNA aptamers is aimed at increasing their thermodynamic stability, and improving affinity and resistance to biodegradation. G-quadruplex DNA aptamers are a family of affinity ligands that form non-canonical DNA assemblies based on a G-tetrads stack. Modification of the quadruplex core is challenging since it can cause complete loss of affinity of the aptamer. On the other hand, increased thermodynamic stability could be a worthy reward. In the current paper, we developed new three- and four-layer modified analogues of the thrombin binding aptamer with high thermal stability, which retain anticoagulant activity against alpha-thrombin. In the modified aptamers, one or two G-tetrads contained non-natural anti-preferred alpha-deoxyguanosines at specific positions. The use of this nucleotide analogue made it possible to control the topology of the modified structures. Due to the presence of non-natural tetrads, we observed some decrease in the anticoagulant activity of the modified aptamers compared to the natural prototype. This negative effect was completely compensated by conjugation of the aptamers with optimized tripeptide sequences. Full article

(This article belongs to the Special Issue Aptamers: Functional-Structural Studies and Biomedical Applications 2.0)

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11 pages, 6301 KiB

Open AccessCommunication

Development of an Anti-Idiotype Aptamer-Based Electrochemical Sensor for a Humanized Therapeutic Antibody Monitoring

by Madoka Nagata, Jinhee Lee, Taro Saito, Kazunori Ikebukuro and Koji Sode

Int. J. Mol. Sci. 2023, 24(6), 5277; https://doi.org/10.3390/ijms24065277 - 9 Mar 2023

Cited by 7 | Viewed by 2306

Abstract

Therapeutic monoclonal antibodies (mAbs) are currently the most effective medicines for a wide range of diseases. Therefore, it is expected that easy and rapid measurement of mAbs will be required to improve their efficacy. Here, we report an anti-idiotype aptamer-based electrochemical sensor for a humanized therapeutic antibody, bevacizumab, based on square wave voltammetry (SWV). With this measurement procedure, we were able to monitor the target mAb within 30 min by employing the anti-idiotype bivalent aptamer modified with a redox probe. A fabricated bevacizumab sensor achieved detection of bevacizumab from 1–100 nM while eliminating the need for free redox probes in the solution. The feasibility of monitoring biological samples was also demonstrated by detecting bevacizumab in the diluted artificial serum, and the fabricated sensor succeeded in detecting the target covering the physiologically relevant concentration range of bevacizumab. Our sensor contributes to ongoing efforts towards therapeutic mAbs monitoring by investigating their pharmacokinetics and improving their treatment efficacy. Full article

(This article belongs to the Special Issue Aptamers: Functional-Structural Studies and Biomedical Applications 2.0)

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19 pages, 4482 KiB

Open AccessArticle

Liquid Crystal@Nanosilver Catalytic Amplification—Aptamer Trimode Biosensor for Trace Pb²⁺

by Yiyi Shu, Sha Li, Chongning Li, Aihui Liang and Zhiliang Jiang

Int. J. Mol. Sci. 2023, 24(3), 2920; https://doi.org/10.3390/ijms24032920 - 2 Feb 2023

Cited by 6 | Viewed by 2387

Abstract

Liquid crystals (LCs) are a very important display material. However, the use of LC, especially LC-loaded nanoparticles, as a catalyst to amplify the analytical signal and coupled with specific aptamer (Apt) as a recognition element to construct a highly sensitive and selective three-mode molecular spectral assay is rarely reported. In this article, five LCs, such as cholesteryl benzoate (CB), were studied by molecular spectroscopy to indicate the liquid crystal nanoparticles in the system, and highly catalytic and stable CB loaded-nanosilver (CB@AgNPs) sol was prepared. The slope procedure was used to study the catalysis of the five LCs and CB@AgNPs on the new indicator reaction between AgNO₃ and sodium formate (Fo) to produce silver nanoparticles (AgNPs) with a strong surface plasmon resonance absorption (Abs) peak at 450 nm, a resonance Rayleigh scattering (RRS) peak at 370 nm and a surface enhanced Raman scattering (SERS) peak at 1618 cm⁻¹ in the presence of molecular probes. By coupling the new CB@AgNPs catalytic indicator reaction with the Apt reaction, a new CB@AgNPs catalytic amplification-SERS/RRS/Abs trimode biosensoring platform was constructed for detecting inorganic pollutants, such as Pb²⁺, Cd²⁺, Hg²⁺ and As³⁺. Full article

(This article belongs to the Special Issue Aptamers: Functional-Structural Studies and Biomedical Applications 2.0)

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19 pages, 2220 KiB

Open AccessArticle

Development of a Molecular Aptamer Beacon Applied to Magnetic-Assisted RNA Extraction for Detection of Dengue and Zika Viruses Using Clinical Samples

by Amanda Gabrielle da Silva, Luiz Ricardo Goulart, Philipp Löffler, Christian Code and Adriana Freitas Neves

Int. J. Mol. Sci. 2022, 23(22), 13866; https://doi.org/10.3390/ijms232213866 - 10 Nov 2022

Cited by 2 | Viewed by 2444

Abstract

Limitations in the detection of cocirculating flaviviruses such as Dengue and Zika lead us to propose the use of aptameric capture of the viral RNA in combination with RT-PCR (APTA-RT-PCR). Aptamers were obtained via SELEX and next-generation sequencing, followed by colorimetric and fluorescent characterizations. An APTA-RT-PCR assay was developed, optimized, and tested against the viral RNAs in 108 serum samples. After selection, sequence APTAZC10 was designed as a bifunctional molecular beacon (APTAZC10-MB), exhibiting affinity for the viral targets. APTA-RT-PCR was able to detect Dengue and Zika RNA in 43% and 8% of samples, respectively. Our results indicate that APTAZC10-MB and APTA-RT-PCR will be useful to improve the detection of Dengue and Zika viruses in a fast molecular assay for the improvement of infectious disease surveillance. Full article

(This article belongs to the Special Issue Aptamers: Functional-Structural Studies and Biomedical Applications 2.0)

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17 pages, 1923 KiB

Open AccessArticle

Sensitive Detection of SARS-CoV-2 Variants Using an Electrochemical Impedance Spectroscopy Based Aptasensor

by Assem Kurmangali, Kanat Dukenbayev and Damira Kanayeva

Int. J. Mol. Sci. 2022, 23(21), 13138; https://doi.org/10.3390/ijms232113138 - 28 Oct 2022

Cited by 7 | Viewed by 2855

Abstract

The global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a threat to public health and a worldwide crisis. This raised the need for quick, effective, and sensitive detection tools to prevent the rapid transmission rate of the infection. Therefore, this study aimed to develop an electrochemical impedance spectroscopy (EIS)-based aptasensor employing an interdigitated gold electrode (IDE) to detect SARS-CoV-2 Spike (S) glycoprotein and viral particles. This allowed us to sensitively detect SARS-CoV-2 S glycoprotein with a limit of detection (LOD) of 0.4 pg/mL in a buffer solution and to obtain a linear increase for concentrations between 0.2 to 0.8 pg/mL with high specificity. The proposed aptasensor also showed a good sensitivity towards the heat-inactivated SARS-CoV-2 variants in a buffer solution, where the Delta, Wuhan, and Alpha variants were captured at a viral titer of 6.45 ± 0.16 × 10³ TCID₅₀/mL, 6.20 × 10⁴ TCID₅₀/mL, and 5.32 ± 0.13 × 10² TCID₅₀/mL, respectively. Furthermore, the detection of SARS-CoV-2 performed in a spiked human nasal fluid provided an LOD of 6.45 ± 0.16 × 10³ TCID₅₀/mL for the Delta variant in a 50 µL sample and a detection time of less than 25 min. Atomic force microscopy images complemented the EIS results in this study, revealing that the surface roughness of the IDE after each modification step increased, which indicates that the target was successfully captured. This label-free EIS-based aptasensor has promising potential for the rapid detection of SARS-CoV-2 in complex clinical samples. Full article

(This article belongs to the Special Issue Aptamers: Functional-Structural Studies and Biomedical Applications 2.0)

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9 pages, 1473 KiB

Open AccessArticle

Nuclease Triggered “Signal-On” and Amplified Fluorescent Sensing of Fumonisin B₁ Incorporating Graphene Oxide and Specific Aptamer

by Xiaodong Guo, Qinqin Qiao, Mengke Zhang and Marie-Laure Fauconnier

Int. J. Mol. Sci. 2022, 23(16), 9024; https://doi.org/10.3390/ijms23169024 - 12 Aug 2022

Cited by 5 | Viewed by 1761

Abstract

Remarkable advancements have been achieved in the development of rapid analytic techniques toward fumonisin B₁ (FB₁) monitoring and even trace levels for food safety in recent years. However, the point-of-care testing for quantitative and accurate FB₁ determination is still challenging. Herein, an innovative aptasensor was established to monitor FB₁ by utilizing graphene oxide (GO) and nuclease-triggered signal enhancement. GO can be utilized as a fluorescence quenching agent toward a fluorophore-modified aptamer, and even as a protectant of the aptamer from nuclease cleavage for subsequent target cycling and signal amplification detection. This proposed sensing strategy exhibited a good linearity for FB₁ determination in the dynamic range from 0.5 to 20 ng mL⁻¹ with a good correlation of R² = 0.995. Its limit of detection was established at 0.15 ng mL⁻¹ (S/N = 3), which was significantly lower than the legal requirements by three orders of magnitude. The interferent study demonstrated that the introduced aptasensor possessed high selectivity for FB₁. Moreover, the aptasensor was successfully applied to the detection of wheat flour samples, and the results were consistent with the classical ELISA method. The rapid response, sensitive and selective analysis, and reliable results of this sensing platform offer a promising opportunity for food mycotoxin control in point-of-care testing. Full article

(This article belongs to the Special Issue Aptamers: Functional-Structural Studies and Biomedical Applications 2.0)

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13 pages, 1596 KiB

Open AccessCommunication

A Polyclonal Aptamer Library for the Specific Binding of the Gut Bacterium Roseburia intestinalis in Mixtures with Other Gut Microbiome Bacteria and Human Stool Samples

by Hu Xing, Yiting Zhang, Markus Krämer, Ann-Kathrin Kissmann, Valerie Amann, Heinz Fabian Raber, Tanja Weil, Kai R. Stieger, Uwe Knippschild, Marius Henkel, Jakob Andersson and Frank Rosenau

Int. J. Mol. Sci. 2022, 23(14), 7744; https://doi.org/10.3390/ijms23147744 - 13 Jul 2022

Cited by 15 | Viewed by 3447

Abstract

Roseburia intestinalis has received attention as a potential probiotic bacterium. Recent studies have demonstrated that changes in its intestinal abundance can cause various diseases, such as obesity, enteritis and atherosclerosis. Probiotic administration or fecal transplantation alter the structure of the intestinal flora, offering possibilities for the prevention and treatment of these diseases. However, current monitoring methods, such as 16S rRNA sequencing, are complex and costly and require specialized personnel to perform the tests, making it difficult to continuously monitor patients during treatment. Hence, the rapid and cost-effective quantification of intestinal bacteria has become an urgent problem to be solved. Aptamers are of emerging interest because their stability, low immunogenicity and ease of modification are attractive properties for a variety of applications. We report a FluCell-SELEX polyclonal aptamer library specific for R. intestinalis isolated after seven evolution rounds, that can bind and label this organism for fluorescence microscopy and binding assays. Moreover, R. intestinalis can be distinguished from other major intestinal bacteria in complex defined mixtures and in human stool samples. We believe that this preliminary evidence opens new avenues towards aptamer-based electronic biosensors as new powerful and inexpensive diagnostic tools for the relative quantitative monitoring of R. intestinalis in gut microbiomes. Full article

(This article belongs to the Special Issue Aptamers: Functional-Structural Studies and Biomedical Applications 2.0)

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Review

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28 pages, 45640 KiB

Open AccessReview

Structural Insights into Protein–Aptamer Recognitions Emerged from Experimental and Computational Studies

by Romualdo Troisi, Nicole Balasco, Ida Autiero, Luigi Vitagliano and Filomena Sica

Int. J. Mol. Sci. 2023, 24(22), 16318; https://doi.org/10.3390/ijms242216318 - 14 Nov 2023

Cited by 4 | Viewed by 2814

Abstract

Aptamers are synthetic nucleic acids that are developed to target with high affinity and specificity chemical entities ranging from single ions to macromolecules and present a wide range of chemical and physical properties. Their ability to selectively bind proteins has made these compounds very attractive and versatile tools, in both basic and applied sciences, to such an extent that they are considered an appealing alternative to antibodies. Here, by exhaustively surveying the content of the Protein Data Bank (PDB), we review the structural aspects of the protein–aptamer recognition process. As a result of three decades of structural studies, we identified 144 PDB entries containing atomic-level information on protein–aptamer complexes. Interestingly, we found a remarkable increase in the number of determined structures in the last two years as a consequence of the effective application of the cryo-electron microscopy technique to these systems. In the present paper, particular attention is devoted to the articulated architectures that protein–aptamer complexes may exhibit. Moreover, the molecular mechanism of the binding process was analyzed by collecting all available information on the structural transitions that aptamers undergo, from their protein-unbound to the protein-bound state. The contribution of computational approaches in this area is also highlighted. Full article

(This article belongs to the Special Issue Aptamers: Functional-Structural Studies and Biomedical Applications 2.0)

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