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Protein and Nucleotide Engineering for Diagnoses and Biosensing

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

Deadline for manuscript submissions: closed (31 May 2022) | Viewed by 43106

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Special Issue Editors

Prof. Dr. Koji Sode

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Guest Editor

Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill (UNC) and North Carolina State University, Chapel Hill, NC, USA
Interests: protein engineering; enzyme engineering; biosensors; biosensing; biomedical engineering; biomolecular engineering
Special Issues, Collections and Topics in MDPI journals

Dr. Takenori Satomura

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Guest Editor

Department of Applied Chemistry and Biotechnology, Faculty of Engineering /Graduate School of Engineering, University of Fukui, Fukui, Japan
Interests: protein
Special Issues, Collections and Topics in MDPI journals

Dr. Wataru Yoshida

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Guest Editor

School of Bioscience and Biotechnology, Graduate School of Bionics, Tokyo University of Technology, Hachioji, Japan
Interests: aptamers; DNA methylation; DNA quadruplex; biosensing
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

The development and innovation of methods and devices for monitoring metabolites, biomarkers, and drugs used for medical treatments are strongly dependent on the availability of the molecular recognition elements—the biosensing molecules. These include proteins such as enzymes, antibodies, binding proteins, and receptors; as well as nucleotides such as DNA aptamers, RNA aptamers, and their mimics such as peptide nucleic acids, which specifically recognize and bind to the target molecules. The recent advancements in the technologies for designing and engineering these molecules, together with the varieties of simulations and prediction tools for engineering proteins/nucleotides, are accelerating the development of engineered biosensing molecules for diagnosis and biosensing technologies, which will provide a new vision of future biosensing technologies.

This Special Issue, “Protein and Nucleotide Engineering for Diagnoses and Biosensing”, aims to provide a summary of the field, to explore recent advances in the discovery, design, and engineering of proteins and nucleotides specifically dedicated for diagnoses and biosensing, and to discuss how we are able to acknowledge them for the development of future biosensing technologies. We invite authors to submit original research articles related to any of these aspects. We also welcome review articles which cover the specific topics in this research area.

Prof. Dr. Koji Sode
Dr. Takenori Satomura
Dr. Wataru Yoshida
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. International Journal of Molecular Sciences is an international peer-reviewed open access semimonthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. There is an Article Processing Charge (APC) for publication in this open access journal. For details about the APC please see here. Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

proteins
enzymes
antibodies
binding proteins
aptamers
biosensors
diagnosis

Published Papers (10 papers)

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Research

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16 pages, 4210 KiB

Open AccessArticle

In-Silico Selection of Aptamer Targeting SARS-CoV-2 Spike Protein

by Yu-Chao Lin, Wen-Yih Chen, En-Te Hwu and Wen-Pin Hu

Int. J. Mol. Sci. 2022, 23(10), 5810; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23105810 - 22 May 2022

Cited by 7 | Viewed by 3038

Abstract

Aptamers are single-stranded, short DNA or RNA oligonucleotides that can specifically bind to various target molecules. To diagnose the infected cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in time, numerous conventional methods are applied for viral detection via the amplification and quantification of DNA or antibodies specific to antigens on the virus. Herein, we generated a large number of mutated aptamer sequences, derived from a known sequence of receptor-binding domain (RBD)-1C aptamer, specific to the RBD of SARS-CoV-2 spike protein (S protein). Structural similarity, molecular docking, and molecular dynamics (MD) were utilized to screen aptamers and characterize the detailed interactions between the selected aptamers and the S protein. We identified two mutated aptamers, namely, RBD-1CM1 and RBD-1CM2, which presented better docking results against the S protein compared with the RBD-1C aptamer. Through the MD simulation, we further confirmed that the RBD-1CM1 aptamer can form the most stable complex with the S protein based on the number of hydrogen bonds formed between the two biomolecules. Based on the experimental data of quartz crystal microbalance (QCM), the RBD-1CM1 aptamer could produce larger signals in mass change and exhibit an improved binding affinity to the S protein. Therefore, the RBD-1CM1 aptamer, which was selected from 1431 mutants, was the best potential candidate for the detection of SARS-CoV-2. The RBD-1CM1 aptamer can be an alternative biological element for the development of SARS-CoV-2 diagnostic testing. Full article

(This article belongs to the Special Issue Protein and Nucleotide Engineering for Diagnoses and Biosensing)

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12 pages, 2656 KiB

Open AccessArticle

Activatable Peptides for Rapid and Simple Visualization of Protease Activity Secreted in Living Cells

by Gae-Baik Kim, Jeong Min Lee, Duc Long Nguyen, Joonseok Lee and Young-Pil Kim

Int. J. Mol. Sci. 2022, 23(3), 1605; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23031605 - 30 Jan 2022

Cited by 2 | Viewed by 1802

Abstract

Activity-based monitoring of cell-secreted proteases has gained significant interest due to the implication of these substances in diverse cellular functions. Here, we demonstrated a cell-based method of monitoring protease activity using fluorescent cell-permeable peptides. The activatable peptide consists of anionic (EEEE), cleavable, and cationic sequences (RRRR) that enable intracellular delivery by matrix metalloproteinase-2 (MMP2), which is secreted by living cancer cells. Compared to HT-29 cells (MMP2-negative), HT-1080 cells (MMP2-positive) showed a strong fluorescence response to the short fluorescent peptide via cell-secreted protease activation. Our approach is expected to find applications for the rapid visualization of protease activity in living cells. Full article

(This article belongs to the Special Issue Protein and Nucleotide Engineering for Diagnoses and Biosensing)

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10 pages, 7891 KiB

Open AccessArticle

Dual-Functional Peroxidase-Copper Phosphate Hybrid Nanoflowers for Sensitive Detection of Biological Thiols

by Xuan Ai Le, Thao Nguyen Le and Moon Il Kim

Int. J. Mol. Sci. 2022, 23(1), 366; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23010366 - 29 Dec 2021

Cited by 5 | Viewed by 1985

Abstract

An effective strategy to detect biological thiols (biothiols), including glutathione (GSH), cysteine (Cys), and homocysteine (Hcy), holds significant incentive since they play vital roles in many cellular processes and are closely related to many diseases. Here, we demonstrated that hybrid nanoflowers composed of crystalline copper phosphate and horseradish peroxidase (HRP) served as a functional unit exhibiting dual catalytic activities of biothiol oxidase and HRP, yielding a cascade reaction system for a sensitive one-pot fluorescent detection of biothiols. The nanoflowers were synthesized through the anisotropic growth of copper phosphate petals coordinated with the amine/amide moieties of HRP, by simply incubating HRP and copper(II) sulfate for three days at room temperature. Copper phosphates within the nanoflowers oxidized target biothiols to generate H₂O₂, which activated the entrapped HRP to oxidize the employed Amplex UltraRed substrate to produce intense fluorescence. Using this strategy, biothiols were selectively and sensitively detected by monitoring the respective fluorescence intensity. This nanoflower-based strategy was also successfully employed for reliable quantification of biothiols present in human serum, demonstrating its great potential for clinical diagnostics. Full article

(This article belongs to the Special Issue Protein and Nucleotide Engineering for Diagnoses and Biosensing)

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Graphical abstract

11 pages, 1616 KiB

Open AccessArticle

Padlock Probe-Based Generation of DNAzymes for the Colorimetric Detection of Antibiotic Resistance Genes

by Rick Conzemius, Ariadne Haunold and Ivan Barišić

Int. J. Mol. Sci. 2021, 22(24), 13654; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms222413654 - 20 Dec 2021

Viewed by 3136

Abstract

The increasing emergence of multidrug- and pan-resistant pathogens requires rapid and cost-efficient diagnostic tools to contain their further spread in healthcare facilities and the environment. The currently established diagnostic technologies are of limited utility for efficient infection control measures because they are either cultivation-based and time-consuming or require sophisticated assays that are expensive. Furthermore, infectious diseases are unfortunately most problematic in countries with low-resource settings in their healthcare systems. In this study, we developed a cost-efficient detection technology that uses G-quadruplex DNAzymes to convert a chromogenic substrate resulting in a color change in the presence of antibiotic resistance genes. The assay is based on padlock probes capable of high-multiplex reactions and targets 27 clinically relevant antibiotic resistance genes associated with sepsis. In addition to an experimental proof-of-principle using synthetic target DNA, the assay was evaluated with multidrug-resistant clinical isolates. Full article

(This article belongs to the Special Issue Protein and Nucleotide Engineering for Diagnoses and Biosensing)

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13 pages, 7660 KiB

Open AccessArticle

Characterization of a Novel Thermostable Dye-Linked l-Lactate Dehydrogenase Complex and Its Application in Electrochemical Detection

by Takenori Satomura, Kohei Uno, Norio Kurosawa, Haruhiko Sakuraba, Toshihisa Ohshima and Shin-ichiro Suye

Int. J. Mol. Sci. 2021, 22(24), 13570; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms222413570 - 17 Dec 2021

Cited by 2 | Viewed by 1947

Abstract

Flavoenzyme dye-linked l-lactate dehydrogenase (Dye-LDH) is primarily involved in energy generation through electron transfer and exhibits potential utility in electrochemical devices. In this study, a gene encoding a Dye-LDH homolog was identified in a hyperthermophilic archaeon, Sulfurisphaera tokodaii. This gene was part of an operon that consisted of four genes that were tandemly arranged in the Sf. tokodaii genome in the following order: stk_16540, stk_16550 (dye-ldh homolog), stk_16560, and stk_16570. This gene cluster was expressed in an archaeal host, Sulfolobus acidocaldarius, and the produced enzyme was purified to homogeneity and characterized. The purified recombinant enzyme exhibited Dye-LDH activity and consisted of two different subunits (products of stk_16540 (α) and stk_16550 (β)), forming a heterohexameric structure (α3β3) with a molecular mass of approximately 253 kDa. Dye-LDH also exhibited excellent stability, retaining full activity upon incubation at 70 °C for 10 min and up to 80% activity after 30 min at 50 °C and pH 6.5–8.0. A quasi-direct electron transfer (DET)-type Dye-LDH was successfully developed by modification of the recombinant enzyme with an artificial redox mediator, phenazine ethosulfate, through amine groups on the enzyme’s surface. This study is the first report describing the development of a quasi-DET-type enzyme by using thermostable Dye-LDH. Full article

(This article belongs to the Special Issue Protein and Nucleotide Engineering for Diagnoses and Biosensing)

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9 pages, 2491 KiB

Open AccessArticle

Highly Efficient Multi-Step Oxidation Bioanode Using Microfluidic Channels

by Tomohiro Komatsu, Kazuki Hishii, Michiko Kimura, Satoshi Amaya, Hiroaki Sakamoto, Eiichiro Takamura, Takenori Satomura and Shin-ichiro Suye

Int. J. Mol. Sci. 2021, 22(24), 13503; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms222413503 - 16 Dec 2021

Viewed by 1924

Abstract

With the rapid decline of fossil fuels, various types of biofuel cells (BFCs) are being developed as an alternative energy source. BFCs based on multi-enzyme cascade reactions are utilized to extract more electrons from substrates. Thus, more power density is obtained from a single molucule of substrate. In the present study, a bioanode that could extract six electrons from a single molecule of L-proline via a three-enzyme cascade reaction was developed and investigated for its possible use in BFCs. These enzymes were immobilized on the electrode to ensure highly efficient electron transfer. Then, oriented immobilization of enzymes was achieved using two types of self-assembled monolayers (SAMs). In addition, a microfluidic system was incorporated to achieve efficient electron transfer. The microfluidic system, in which the electrodes were arranged in a tooth-shaped comb, allowed for substrates to be supplied continuously to the cascade, which resulted in smooth electron transfer. Finally, we developed a high-performance bioanode which resulted in the accumulation of higher current density compared to that of a gold disc electrode (205.8 μA cm⁻²: approximately 187 times higher). This presents an opportunity for using the bioanode to develop high-performance BFCs in the future. Full article

(This article belongs to the Special Issue Protein and Nucleotide Engineering for Diagnoses and Biosensing)

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15 pages, 735 KiB

Open AccessArticle

Quantitative Estimate Index for Early-Stage Screening of Compounds Targeting Protein-Protein Interactions

by Takatsugu Kosugi and Masahito Ohue

Int. J. Mol. Sci. 2021, 22(20), 10925; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms222010925 - 10 Oct 2021

Cited by 11 | Viewed by 4397

Abstract

Drug-likeness quantification is useful for screening drug candidates. Quantitative estimates of drug-likeness (QED) are commonly used to assess quantitative drug efficacy but are not suitable for screening compounds targeting protein-protein interactions (PPIs), which have recently gained attention. Therefore, we developed a quantitative estimate index for compounds targeting PPIs (QEPPI), specifically for early-stage screening of PPI-targeting compounds. QEPPI is an extension of the QED method for PPI-targeting drugs that models physicochemical properties based on the information available for drugs/compounds, specifically those reported to act on PPIs. FDA-approved drugs and compounds in iPPI-DB, which comprise PPI inhibitors and stabilizers, were evaluated using QEPPI. The results showed that QEPPI is more suitable than QED for early screening of PPI-targeting compounds. QEPPI was also considered an extended concept of the “Rule-of-Four” (RO4), a PPI inhibitor index. We evaluated the discriminatory performance of QEPPI and RO4 for datasets of PPI-target compounds and FDA-approved drugs using F-score and other indices. The F-scores of RO4 and QEPPI were 0.451 and 0.501, respectively. QEPPI showed better performance and enabled quantification of drug-likeness for early-stage PPI drug discovery. Hence, it can be used as an initial filter to efficiently screen PPI-targeting compounds. Full article

(This article belongs to the Special Issue Protein and Nucleotide Engineering for Diagnoses and Biosensing)

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12 pages, 3497 KiB

Open AccessArticle

One-Step Surface Immobilization of Protein A on Hydrogel Nanofibers by Core-Shell Electrospinning for Capturing Antibodies

by Chihiro Naganuma, Kosuke Moriyama, Shin-ichiro Suye and Satoshi Fujita

Int. J. Mol. Sci. 2021, 22(18), 9857; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22189857 - 12 Sep 2021

Cited by 2 | Viewed by 2286

Abstract

Nanofibers (NFs) are potential candidates as filter materials for affinity separation owing to their high liquid permeability based on their high porosity. Multiple and complex processes were conventionally performed to immobilize proteins for modifying NF surfaces. A simple method must be developed to immobilize proteins without impairing their biological activity. Herein, we succeeded in fabricating NFs with a core of cellulose acetate and a shell of hydrophilic polyvinyl alcohol immobilized with staphylococcal recombinant protein A by a one-step process based on core-shell electrospinning. A total of 12.9 mg/cm³ of antibody was captured in the fiber shell through high affinity with protein A immobilized in an aqueous environment of the hydrogel. The maximum adsorption site and dissociation constant evaluated by the Langmuir model were 87.8 µg and 1.37 µmol/L, respectively. The fiber sheet withstood triplicate use. Thus, our NF exhibited high potential as a material for membrane chromatography. Full article

(This article belongs to the Special Issue Protein and Nucleotide Engineering for Diagnoses and Biosensing)

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Review

Jump to: Research

11 pages, 2111 KiB

Open AccessReview

Fabrication of Fragment Antibody–Enzyme Complex as a Sensing Element for Immunosensing

by Miho Oda and Ryutaro Asano

Int. J. Mol. Sci. 2022, 23(3), 1335; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23031335 - 25 Jan 2022

Cited by 4 | Viewed by 2743

Abstract

Antibody–enzyme complexes (AECs) are ideal molecular recognition elements for immunosensing applications. One molecule possesses both a binding ability to specific targets and catalytic activity to gain signals, particularly oxidoreductases, which can be integrated into rapid and sensitive electrochemical measurements. The development of AECs using fragment antibodies rather than intact antibodies, such as immunoglobulin G (IgG), has attracted attention for overcoming the ethical and cost issues associated with the production of intact antibodies. Conventionally, chemical conjugation has been used to fabricate AECs; however, controlling stoichiometric conjugation using this method is difficult. To prepare homogeneous AECs, methods based on direct fusion and enzymatic conjugation have been developed, and more convenient methods using Catcher/Tag systems as coupling modules have been reported. In this review, we summarize the methods for fabricating AECs using fragment antibodies developed for sensing applications and discuss the advantages and disadvantages of each method. Full article

(This article belongs to the Special Issue Protein and Nucleotide Engineering for Diagnoses and Biosensing)

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29 pages, 1395 KiB

Open AccessReview

Endometriosis: Epidemiology, Classification, Pathogenesis, Treatment and Genetics (Review of Literature)

by Beata Smolarz, Krzysztof Szyłło and Hanna Romanowicz

Int. J. Mol. Sci. 2021, 22(19), 10554; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms221910554 - 29 Sep 2021

Cited by 116 | Viewed by 18466

Abstract

Endometriosis is a “mysterious” disease and its exact cause has not yet been established. Among the etiological factors, congenital, environmental, epigenetic, autoimmune and allergic factors are listed. It is believed that the primary mechanism of the formation of endometriosis foci is retrograde menstruation, i.e., the passage of menstrual blood through the fallopian tubes into the peritoneal cavity and implantation of exfoliated endometrial cells. However, since this mechanism is also observed in healthy women, other factors must also be involved in the formation of endometriosis foci. Endometriosis is in many women the cause of infertility, chronic pain and the deterioration of the quality of life. It also represents a significant financial burden on health systems. The article presents a review of the literature on endometriosis—a disease affecting women throughout the world. Full article

(This article belongs to the Special Issue Protein and Nucleotide Engineering for Diagnoses and Biosensing)

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