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4 pages, 173 KiB

Open AccessEditorial

Nucleic Acids Chemistry and Engineering: Special Issue on Nucleic Acid Conjugates for Biotechnological Applications

by Tamaki Endoh, Eriks Rozners and Takashi Ohtsuki

Appl. Sci. 2021, 11(8), 3594; https://doi.org/10.3390/app11083594 - 16 Apr 2021

Cited by 1 | Viewed by 1212

Abstract

Nucleic acids not only store genetic information in their primary sequence but also exhibit biological functions through the formation of their unique structures [...] Full article

(This article belongs to the Special Issue Nucleic Acids Conjugates for Biotechnological Applications)

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16 pages, 3443 KiB

Open AccessFeature PaperArticle

An RNA Triangle with Six Ribozyme Units Can Promote a Trans-Splicing Reaction through Trimerization of Unit Ribozyme Dimers

by Junya Akagi, Takahiro Yamada, Kumi Hidaka, Yoshihiko Fujita, Hirohide Saito, Hiroshi Sugiyama, Masayuki Endo, Shigeyoshi Matsumura and Yoshiya Ikawa

Appl. Sci. 2021, 11(6), 2583; https://doi.org/10.3390/app11062583 - 14 Mar 2021

Cited by 3 | Viewed by 2233

Abstract

Ribozymes are catalytic RNAs that are attractive platforms for the construction of nanoscale objects with biological functions. We designed a dimeric form of the Tetrahymena group I ribozyme as a unit structure in which two ribozymes were connected in a tail-to-tail manner with [...] Read more.

Ribozymes are catalytic RNAs that are attractive platforms for the construction of nanoscale objects with biological functions. We designed a dimeric form of the Tetrahymena group I ribozyme as a unit structure in which two ribozymes were connected in a tail-to-tail manner with a linker element. We introduced a kink-turn motif as a bent linker element of the ribozyme dimer to design a closed trimer with a triangular shape. The oligomeric states of the resulting ribozyme dimers (kUrds) were analyzed biochemically and observed directly by atomic force microscopy (AFM). Formation of kUrd oligomers also triggered trans-splicing reactions, which could be monitored with a reporter system to yield a fluorescent RNA aptamer as the trans-splicing product. Full article

(This article belongs to the Special Issue Nucleic Acids Conjugates for Biotechnological Applications)

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15 pages, 2886 KiB

Open AccessFeature PaperArticle

Novel Lipid-Oligonucleotide Conjugates Containing Long-Chain Sulfonyl Phosphoramidate Groups: Synthesis and Biological Properties

by Alina Derzhalova, Oleg Markov, Alesya Fokina, Yasuo Shiohama, Timofei Zatsepin, Masayuki Fujii, Marina Zenkova and Dmitry Stetsenko

Appl. Sci. 2021, 11(3), 1174; https://doi.org/10.3390/app11031174 - 27 Jan 2021

Cited by 10 | Viewed by 3784

Abstract

New lipid conjugates of DNA and RNA incorporating one to four [(4-dodecylphenyl)sulfonyl]phosphoramidate or (hexadecylsulfonyl)phosphoramidate groups at internucleotidic positions near the 3′ or 5′-end were synthesized and characterized. Low cytotoxicity of the conjugates and their ability to be taken up into cells without transfection [...] Read more.

New lipid conjugates of DNA and RNA incorporating one to four [(4-dodecylphenyl)sulfonyl]phosphoramidate or (hexadecylsulfonyl)phosphoramidate groups at internucleotidic positions near the 3′ or 5′-end were synthesized and characterized. Low cytotoxicity of the conjugates and their ability to be taken up into cells without transfection agents were demonstrated. Lipid-conjugated siRNAs targeting repulsive guidance molecules a (RGMa) have shown a comparable gene silencing activity in PK-59 cells to unmodified control siRNA when delivered into the cells via Lipofectamine mediated transfection. Full article

(This article belongs to the Special Issue Nucleic Acids Conjugates for Biotechnological Applications)

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10 pages, 1450 KiB

Open AccessFeature PaperArticle

RNA-Peptide Conjugation through an Efficient Covalent Bond Formation

by Shun Nakano, Taiki Seko, Zhengxiao Zhang and Takashi Morii

Appl. Sci. 2020, 10(24), 8920; https://doi.org/10.3390/app10248920 - 14 Dec 2020

Cited by 3 | Viewed by 3439

Abstract

Many methods for modification of an oligonucleotide with a peptide have been developed to apply for the therapeutic and diagnostic applications or for the assembly of nanostructure. We have developed a method for the construction of receptor-based fluorescent sensors and catalysts using the [...] Read more.

Many methods for modification of an oligonucleotide with a peptide have been developed to apply for the therapeutic and diagnostic applications or for the assembly of nanostructure. We have developed a method for the construction of receptor-based fluorescent sensors and catalysts using the ribonucleopeptide (RNP) as a scaffold. Formation of a covalent linkage between the RNA and the peptide subunit of RNP improved its stability, thereby expanding the application of functional RNPs. A representative method was applied for the formation of Schiff base or dihydroxy-morpholino linkage between a dialdehyde group at the 3′-end of sugar-oxidized RNA and a hydrazide group introduced at the C-terminal of a peptide subunit through a flexible peptide linker. In this report, we investigated effects of the solution pH and contribution of the RNA and peptide subunits to the conjugation reaction by using RNA and peptide mutants. The reaction yield reached 90% at a wide range of solution pH with reaction within 3 h. The efficient reaction was mainly supported by the electrostatic interaction between the RNA subunit and the cationic peptide subunit of the RNP scaffold. Formation of the RNP complex was verified to efficiently promote the reaction for construction of the RNA-peptide conjugate. Full article

(This article belongs to the Special Issue Nucleic Acids Conjugates for Biotechnological Applications)

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9 pages, 2303 KiB

Open AccessFeature PaperCommunication

Bifunctional Aptamer Drug Carrier Enabling Selective and Efficient Incorporation of an Approved Anticancer Drug Irinotecan to Fibrin Gels

by Hiroto Fujita, Yuka Kataoka and Masayasu Kuwahara

Appl. Sci. 2020, 10(23), 8755; https://doi.org/10.3390/app10238755 - 07 Dec 2020

Cited by 1 | Viewed by 2146

Abstract

We have previously developed a bifunctional aptamer (bApt) binding to both human thrombin and camptothecin derivative (CPT1), and showed that bApt acts as a drug carrier under the phenomenon named selective oligonucleotide entrapment in fibrin polymers (SOEF), which enables efficient enrichment of CPT1 [...] Read more.

We have previously developed a bifunctional aptamer (bApt) binding to both human thrombin and camptothecin derivative (CPT1), and showed that bApt acts as a drug carrier under the phenomenon named selective oligonucleotide entrapment in fibrin polymers (SOEF), which enables efficient enrichment of CPT1 into fibrin gels, resulting in significant inhibition of tumor cell growth. However, although the derivative CPT1 exhibits anticancer activity, it is not an approved drug. In this study, we evaluated the binding properties of bApt to irinotecan, a camptothecin analog commonly used for anticancer drug therapy, in addition to unmodified camptothecin (CPT). Furthermore, we have revealed that irinotecan binds to bApt like CPT1 and is selectively concentrated on fibrin gels formed around the tumor cells under the SOEF phenomenon to suppress cell proliferation. Full article

(This article belongs to the Special Issue Nucleic Acids Conjugates for Biotechnological Applications)

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9 pages, 1237 KiB

Open AccessFeature PaperCommunication

Investigation of the Characteristics of NLS-PNA: Influence of NLS Location on Invasion Efficiency

by Yuichiro Aiba, Gerardo Urbina, Masanari Shibata and Osami Shoji

Appl. Sci. 2020, 10(23), 8663; https://doi.org/10.3390/app10238663 - 03 Dec 2020

Cited by 6 | Viewed by 2753

Abstract

Peptide nucleic acid can recognise sequences in double-stranded DNA (dsDNA) through the formation of a double-duplex invasion complex. This double-duplex invasion is a promising method for the recognition of dsDNA in cellula because peptide nucleic acid (PNA) invasion does not require the prior [...] Read more.

Peptide nucleic acid can recognise sequences in double-stranded DNA (dsDNA) through the formation of a double-duplex invasion complex. This double-duplex invasion is a promising method for the recognition of dsDNA in cellula because peptide nucleic acid (PNA) invasion does not require the prior denaturation of dsDNA. To increase its applicability, we developed PNAs modified with a nuclear localisation signal (NLS) peptide. In this study, the characteristics of NLS-modified PNAs were investigated for the future design of novel peptide-modified PNAs. Full article

(This article belongs to the Special Issue Nucleic Acids Conjugates for Biotechnological Applications)

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10 pages, 2901 KiB

Open AccessFeature PaperArticle

Encapsulation of mRNA into Artificial Viral Capsids via Hybridization of a β-Annulus-dT₂₀ Conjugate and the Poly(A) Tail of mRNA

by Yoko Nakamura, Yuki Sato, Hiroshi Inaba, Takashi Iwasaki and Kazunori Matsuura

Appl. Sci. 2020, 10(22), 8004; https://doi.org/10.3390/app10228004 - 12 Nov 2020

Cited by 11 | Viewed by 4133

Abstract

Messenger RNA (mRNA) drugs have attracted considerable attention as promising tools with many therapeutic applications. The efficient delivery of mRNA drugs using non-viral materials is currently being explored. We demonstrate a novel concept where mCherry mRNA bearing a poly(A) tail is encapsulated into [...] Read more.

Messenger RNA (mRNA) drugs have attracted considerable attention as promising tools with many therapeutic applications. The efficient delivery of mRNA drugs using non-viral materials is currently being explored. We demonstrate a novel concept where mCherry mRNA bearing a poly(A) tail is encapsulated into capsids co-assembled from viral β-annulus peptides bearing a 20-mer oligothymine (dT₂₀) at the N-terminus and unmodified peptides via hybridization of dT₂₀ and poly(A). Dynamic light scattering measurements and transmission electron microscopy images of the mRNA-encapsulated capsids show the formation of spherical assemblies of approximately 50 nm. The encapsulated mRNA shows remarkable ribonuclease resistance. Further, modification by a cell-penetrating peptide (His16) on the capsid enables the intracellular expression of mCherry of encapsulated mRNA. Full article

(This article belongs to the Special Issue Nucleic Acids Conjugates for Biotechnological Applications)

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14 pages, 3310 KiB

Open AccessFeature PaperArticle

Design of the Crosslinking Reactions for Nucleic Acids-Binding Protein and Evaluation of the Reactivity

by Kenta Odaira, Ken Yamada, Shogo Ishiyama, Hidenori Okamura and Fumi Nagatsugi

Appl. Sci. 2020, 10(21), 7709; https://doi.org/10.3390/app10217709 - 30 Oct 2020

Cited by 2 | Viewed by 2474

Abstract

Selective chemical reactions of biomolecules are some of the important tools for investigations by biological studies. We have developed the selective crosslinking reactions to form covalent bonds to DNA or RNA using crosslinking oligonucleotides (CFO) bearing reactive bases. In this study, we designed [...] Read more.

Selective chemical reactions of biomolecules are some of the important tools for investigations by biological studies. We have developed the selective crosslinking reactions to form covalent bonds to DNA or RNA using crosslinking oligonucleotides (CFO) bearing reactive bases. In this study, we designed the cross-linkable 4-amino-6-oxo-2-vinyltriazine derivative with an acyclic linker (acyAOVT) to react with the nucleic acids-binding protein based on our previous results. We hypothesized that the acyAOVT base would form a stable base pair with guanine by three hydrogen bonds at the positions of the vinyl group in the duplex DNA major groove, and the vinyl group can react with the nucleophilic species in the proximity, for example, the cysteine or lysine residue in the nucleic acids-binding protein. The synthesized oligonucleotides bearing the acyAOVT derivative showed a higher reactivity than that of the corresponding pyrimidine derivative without one nitrogen. The duplex containing acyAOVT-guanine (G) formed complexes with Hha1 DNMT even in the presence of 2-mercaptoethanol. We expect that our system will provide a useful tool for the molecular study of nucleic acids-binding proteins. Full article

(This article belongs to the Special Issue Nucleic Acids Conjugates for Biotechnological Applications)

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10 pages, 2707 KiB

Open AccessFeature PaperArticle

Construction of an Enzymatically-Conjugated DNA Aptamer–Protein Hybrid Molecule for Use as a BRET-Based Biosensor

by Masayasu Mie, Rena Hirashima, Yasumasa Mashimo and Eiry Kobatake

Appl. Sci. 2020, 10(21), 7646; https://doi.org/10.3390/app10217646 - 29 Oct 2020

Cited by 5 | Viewed by 2389

Abstract

DNA-protein conjugates are useful molecules for construction of biosensors. Herein, we report the development of an enzymatically-conjugated DNA aptamer–protein hybrid molecule for use as a bioluminescence resonance energy transfer (BRET)-based biosensor. DNA aptamers were enzymatically conjugated to a fusion protein via the catalytic [...] Read more.

DNA-protein conjugates are useful molecules for construction of biosensors. Herein, we report the development of an enzymatically-conjugated DNA aptamer–protein hybrid molecule for use as a bioluminescence resonance energy transfer (BRET)-based biosensor. DNA aptamers were enzymatically conjugated to a fusion protein via the catalytic domain of porcine circovirus type 2 replication initiation protein (PCV2 Rep) comprising residues 14–109 (tpRep), which was truncated from the full catalytic domain of PCV2 Rep comprising residues 1–116 by removing the flexible regions at the N- and C-terminals. For development of a BRET-based biosensor, we constructed a fusion protein in which tpRep was positioned between NanoLuc luciferase and a fluorescent protein and conjugated to single-stranded DNA aptamers that specifically bind to either thrombin or lysozyme. We demonstrated that the BRET ratios depended on the concentration of the target molecules. Full article

(This article belongs to the Special Issue Nucleic Acids Conjugates for Biotechnological Applications)

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9 pages, 1356 KiB

Open AccessFeature PaperArticle

Effective RNA Regulation by Combination of Multiple Programmable RNA-Binding Proteins

by Misaki Sugimoto, Akiyo Suda, Shiroh Futaki and Miki Imanishi

Appl. Sci. 2020, 10(19), 6803; https://doi.org/10.3390/app10196803 - 28 Sep 2020

Cited by 3 | Viewed by 2147

Abstract

RNAs play important roles in gene expression through translation and RNA splicing. Regulation of specific RNAs is useful to understand and manipulate specific transcripts. Pumilio and fem-3 mRNA-binding factor (PUF) proteins, programmable RNA-binding proteins, are promising tools for regulating specific RNAs by fusing [...] Read more.

RNAs play important roles in gene expression through translation and RNA splicing. Regulation of specific RNAs is useful to understand and manipulate specific transcripts. Pumilio and fem-3 mRNA-binding factor (PUF) proteins, programmable RNA-binding proteins, are promising tools for regulating specific RNAs by fusing them with various functional domains. The key question is: How can PUF-based molecular tools efficiently regulate RNA functions? Here, we show that the combination of multiple PUF proteins, compared to using a single PUF protein, targeting independent RNA sequences at the 3′ untranslated region (UTR) of a target transcript caused cooperative effects to regulate the function of the target RNA by luciferase reporter assays. It is worth noting that a higher efficacy was achieved with smaller amounts of each PUF expression vector introduced into the cells compared to using a single PUF protein. This strategy not only efficiently regulates target RNA functions but would also be effective in reducing off-target effects due to the low doses of each expression vector. Full article

(This article belongs to the Special Issue Nucleic Acids Conjugates for Biotechnological Applications)

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9 pages, 1729 KiB

Open AccessFeature PaperArticle

Chloro-Substituted Naphthyridine Derivative and Its Conjugate with Thiazole Orange for Highly Selective Fluorescence Sensing of an Orphan Cytosine in the AP Site-Containing Duplexes

by Chun-xia Wang, Yusuke Sato, Takashi Sugimoto, Norio Teramae and Seiichi Nishizawa

Appl. Sci. 2020, 10(12), 4133; https://doi.org/10.3390/app10124133 - 16 Jun 2020

Cited by 4 | Viewed by 1874

Abstract

Fluorescent probes with the binding selectivity to specific structures in DNAs or RNAs have gained much attention as useful tools for the study of nucleic acid functions. Here, chloro-substituted 2-amino-5,7-dimethyl-1,8-naphthyridine (ClNaph) was developed as a strong and highly selective binder for target orphan [...] Read more.

Fluorescent probes with the binding selectivity to specific structures in DNAs or RNAs have gained much attention as useful tools for the study of nucleic acid functions. Here, chloro-substituted 2-amino-5,7-dimethyl-1,8-naphthyridine (ClNaph) was developed as a strong and highly selective binder for target orphan cytosine opposite an abasic (AP) site in the DNA duplexes. ClNaph was then conjugated with thiazole orange (TO) via an alkyl spacer (ClNaph–TO) to design a light-up probe for the detection of cytosine-related mutations in target DNA. In addition, we found the useful binding and fluorescence signaling of the ClNaph–TO conjugate to target C in AP site-containing DNA/RNA hybrid duplexes with a view toward sequence analysis of microRNAs. Full article

(This article belongs to the Special Issue Nucleic Acids Conjugates for Biotechnological Applications)

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