Molecules

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13 pages, 1964 KiB

Open AccessArticle

Site Selective Antibody-Oligonucleotide Conjugation via Microbial Transglutaminase

by Ian J. Huggins, Carlos A. Medina, Aaron D. Springer, Arjen van den Berg, Satish Jadhav, Xianshu Cui and Steven F. Dowdy

Molecules 2019, 24(18), 3287; https://doi.org/10.3390/molecules24183287 - 10 Sep 2019

Cited by 18 | Viewed by 8415

Abstract

Nucleic Acid Therapeutics (NATs), including siRNAs and AntiSense Oligonucleotides (ASOs), have great potential to drug the undruggable genome. Targeting siRNAs and ASOs to specific cell types of interest has driven dramatic improvement in efficacy and reduction in toxicity. Indeed, conjugation of tris-GalNAc to [...] Read more.

Nucleic Acid Therapeutics (NATs), including siRNAs and AntiSense Oligonucleotides (ASOs), have great potential to drug the undruggable genome. Targeting siRNAs and ASOs to specific cell types of interest has driven dramatic improvement in efficacy and reduction in toxicity. Indeed, conjugation of tris-GalNAc to siRNAs and ASOs has shown clinical efficacy in targeting diseases driven by liver hepatocytes. However, targeting non-hepatic diseases with oligonucleotide therapeutics has remained problematic for several reasons, including targeting specific cell types and endosomal escape. Monoclonal antibody (mAb) targeting of siRNAs and ASOs has the potential to deliver these drugs to a variety of specific cell and tissue types. However, most conjugation strategies rely on random chemical conjugation through lysine or cysteine residues resulting in conjugate heterogeneity and a distribution of Drug:Antibody Ratios (DAR). To produce homogeneous DAR-2 conjugates with two siRNAs per mAb, we developed a novel two-step conjugation procedure involving microbial transglutaminase (MTGase) tagging of the antibody C-terminus with an azide-functionalized linker peptide that can be subsequently conjugated to dibenzylcyclooctyne (DBCO) bearing oligonucleotides through azide-alkyne cycloaddition. Antibody-siRNA (and ASO) conjugates (ARCs) produced using this strategy are soluble, chemically defined targeted oligonucleotide therapeutics that have the potential to greatly increase the number of targetable cell types. Full article

(This article belongs to the Special Issue Molecular Tools for Nucleic Acid Manipulation for Biological Intervention)

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12 pages, 1988 KiB

Open AccessArticle

Factors Influencing the Activity of Nanozymes in the Cleavage of an RNA Model Substrate

by Joanna Czescik, Susanna Zamolo, Tamis Darbre, Fabrizio Mancin and Paolo Scrimin

Molecules 2019, 24(15), 2814; https://doi.org/10.3390/molecules24152814 - 1 Aug 2019

Cited by 16 | Viewed by 3902

Abstract

A series of 2-nm gold nanoparticles passivated with different thiols all featuring at least one triazacyclonanone-Zn(II) complex and different flanking units (a second Zn(II) complex, a triethyleneoxymethyl derivative or a guanidinium of arginine of a peptide) were prepared and studied for their efficiency [...] Read more.

A series of 2-nm gold nanoparticles passivated with different thiols all featuring at least one triazacyclonanone-Zn(II) complex and different flanking units (a second Zn(II) complex, a triethyleneoxymethyl derivative or a guanidinium of arginine of a peptide) were prepared and studied for their efficiency in the cleavage of the RNA-model substrate 2-hydroxypropyl-p-nitrophenyl phosphate. The source of catalysis for each of them was elucidated from the kinetic analysis (Michaelis–Menten profiles, pH dependence and kinetic isotope effect). The data indicated that two different mechanisms were operative: One involving two Zn(II) complexes and the other one involving a single Zn(II) complex and a flanking guanidinium cation. The mechanism based on a dinuclear catalytic site appeared more efficient than the one based on the cooperativity between a metal complex and a guanidinium. Full article

(This article belongs to the Special Issue Molecular Tools for Nucleic Acid Manipulation for Biological Intervention)

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17 pages, 1918 KiB

Open AccessArticle

N1-Propargylguanosine Modified mRNA Cap Analogs: Synthesis, Reactivity, and Applications to the Study of Cap-Binding Proteins

by Michal Kopcial, Blazej A. Wojtczak, Renata Kasprzyk, Joanna Kowalska and Jacek Jemielity

Molecules 2019, 24(10), 1899; https://doi.org/10.3390/molecules24101899 - 17 May 2019

Cited by 6 | Viewed by 4712

Abstract

The mRNA 5′ cap consists of N7-methylguanosine bound by a 5′,5′-triphosphate bridge to the first nucleotide of the transcript. The cap interacts with various specific proteins and participates in all key mRNA-related processes, which may be of therapeutic relevance. There is a growing [...] Read more.

The mRNA 5′ cap consists of N7-methylguanosine bound by a 5′,5′-triphosphate bridge to the first nucleotide of the transcript. The cap interacts with various specific proteins and participates in all key mRNA-related processes, which may be of therapeutic relevance. There is a growing demand for new biophysical and biochemical methods to study cap–protein interactions and identify the factors which inhibit them. The development of such methods can be aided by the use of properly designed fluorescent molecular probes. Herein, we synthesized a new class of m⁷Gp₃G cap derivatives modified with an alkyne handle at the N1-position of guanosine and, using alkyne-azide cycloaddition, we functionalized them with fluorescent tags to obtain potential probes. The cap derivatives and probes were evaluated in the context of two cap-binding proteins, eukaryotic translation initiation factor (eIF4E) and decapping scavenger (DcpS). Biochemical and biophysical studies revealed that N1-propargyl moiety did not significantly disturb cap–protein interaction. The fluorescent properties of the probes turned out to be in line with microscale thermophoresis (MST)-based binding assays. Full article

(This article belongs to the Special Issue Molecular Tools for Nucleic Acid Manipulation for Biological Intervention)

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16 pages, 3777 KiB

Open AccessArticle

PEG-OligoRNA Hybridization of mRNA for Developing Sterically Stable Lipid Nanoparticles toward In Vivo Administration

by Shota Kurimoto, Naoto Yoshinaga, Kazunori Igarashi, Yu Matsumoto, Horacio Cabral and Satoshi Uchida

Molecules 2019, 24(7), 1303; https://doi.org/10.3390/molecules24071303 - 3 Apr 2019

Cited by 20 | Viewed by 6716

Abstract

Lipid nanoparticles (LNPs) exhibit high potential as carriers of messenger RNA (mRNA). However, the arduous preparation process of mRNA-loaded LNPs remains a huge obstacle for their widespread clinical application. Herein, we tackled this issue by mRNA PEGylation through hybridization with polyethylene glycol (PEG)-conjugated [...] Read more.

Lipid nanoparticles (LNPs) exhibit high potential as carriers of messenger RNA (mRNA). However, the arduous preparation process of mRNA-loaded LNPs remains a huge obstacle for their widespread clinical application. Herein, we tackled this issue by mRNA PEGylation through hybridization with polyethylene glycol (PEG)-conjugated RNA oligonucleotides (PEG-OligoRNAs). Importantly, mRNA translational activity was preserved even after hybridization of 20 PEG-OligoRNAs per mRNA. The straightforward mixing of the PEGylated mRNA with lipofectamine LTX, a commercial lipid-based carrier, just by pipetting in aqueous solution, allowed the successful preparation of mRNA-loaded LNPs with a diameter below 100 nm, whereas the use of non-PEGylated mRNA provided large aggregates above 100- and 1000-nm. In vivo, LNPs prepared from PEG-OligoRNA-hybridized mRNA exhibited high structural stability in biological milieu, without forming detectable aggregates in mouse blood after intravenous injection. In contrast, LNPs from non-PEGylated mRNA formed several micrometer-sized aggregates in blood, leading to rapid clearance from blood circulation and deposition of the aggregates in lung capillaries. Our strategy of mRNA PEGylation was also versatile to prevent aggregation of another type of mRNA-loaded LNP, DOTAP/Chol liposomes. Together, our approach provides a simple and robust preparation method to LNPs for in vivo application. Full article

(This article belongs to the Special Issue Molecular Tools for Nucleic Acid Manipulation for Biological Intervention)

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13 pages, 1131 KiB

Open AccessArticle

Oligonucleotide–Palladacycle Conjugates as Splice-Correcting Agents

by Madhuri Hande, Osama Saher, Karin E. Lundin, C. I. Edvard Smith, Rula Zain and Tuomas Lönnberg

Molecules 2019, 24(6), 1180; https://doi.org/10.3390/molecules24061180 - 26 Mar 2019

Cited by 10 | Viewed by 5052

Abstract

2’-O-Methylribo phosphorothioate oligonucleotides incorporating cyclopalladated benzylamine conjugate groups at their 5’-termini have been prepared and their ability to hybridize with a designated target sequence was assessed by conventional UV melting experiments. The oligonucleotides were further examined in splice-switching experiments in human [...] Read more.

2’-O-Methylribo phosphorothioate oligonucleotides incorporating cyclopalladated benzylamine conjugate groups at their 5’-termini have been prepared and their ability to hybridize with a designated target sequence was assessed by conventional UV melting experiments. The oligonucleotides were further examined in splice-switching experiments in human cervical cancer (HeLa Luc/705), human liver (HuH7_705), and human osteosarcoma (U-2 OS_705) reporter cell lines. Melting temperatures of duplexes formed by the modified oligonucleotides were approximately 5 °C lower than melting temperatures of the respective unmodified duplexes. The cyclopalladated oligonucleotides functioned as splice-correcting agents in the HeLa Luc/705 cell line somewhat more efficiently than their unmodified counterparts. Furthermore, the introduction of this chemical modification did not induce toxicity in cells. These results demonstrate the feasibility of using covalently metalated oligonucleotides as therapeutic agents. Full article

(This article belongs to the Special Issue Molecular Tools for Nucleic Acid Manipulation for Biological Intervention)

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11 pages, 1853 KiB

Open AccessArticle

Oligonucleotide Binding to Non-B-DNA in MYC

by Tea Umek, Karin Sollander, Helen Bergquist, Jesper Wengel, Karin E. Lundin, C.I. Edvard Smith and Rula Zain

Molecules 2019, 24(5), 1000; https://doi.org/10.3390/molecules24051000 - 12 Mar 2019

Cited by 4 | Viewed by 4524

Abstract

MYC, originally named c-myc, is an oncogene deregulated in many different forms of cancer. Translocation of the MYC gene to an immunoglobulin gene leads to an overexpression and the development of Burkitt’s lymphoma (BL). Sporadic BL constitutes one subgroup where [...] Read more.

MYC, originally named c-myc, is an oncogene deregulated in many different forms of cancer. Translocation of the MYC gene to an immunoglobulin gene leads to an overexpression and the development of Burkitt’s lymphoma (BL). Sporadic BL constitutes one subgroup where one of the translocation sites is located at the 5’-vicinity of the two major MYC promoters P₁ and P₂. A non-B-DNA forming sequence within this region has been reported with the ability to form an intramolecular triplex (H-DNA) or a G-quadruplex. We have examined triplex formation at this site first by using a 17 bp triplex-forming oligonucleotide (TFO) and a double strand DNA (dsDNA) target corresponding to the MYC sequence. An antiparallel purine-motif triplex was detected using electrophoretic mobility shift assay. Furthermore, we probed for H-DNA formation using the BQQ-OP based triplex-specific cleavage assay, which indicated the formation of the structure in the supercoiled plasmid containing the corresponding region of the MYC promoter. Targeting non-B-DNA structures has therapeutic potential; therefore, we investigated their influence on strand-invasion of anti-gene oligonucleotides (ON)s. We show that in vitro, non-B-DNA formation at the vicinity of the ON target site facilitates dsDNA strand-invasion of the anti-gene ONs. Full article

(This article belongs to the Special Issue Molecular Tools for Nucleic Acid Manipulation for Biological Intervention)

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11 pages, 2832 KiB

Open AccessArticle

Site-Specific Cleavage of RNAs Derived from the PIM1 3′-UTR by a Metal-Free Artificial Ribonuclease

by Felix Zellmann, Laura Thomas, Ute Scheffer, Roland K. Hartmann and Michael W. Göbel

Molecules 2019, 24(4), 807; https://doi.org/10.3390/molecules24040807 - 23 Feb 2019

Cited by 10 | Viewed by 4860

Abstract

Oligonucleotide conjugates of tris(2-aminobenzimidazole) have been reported previously to cleave complementary RNA strands with high levels of sequence and site specificity. The RNA substrates used in these studies were oligonucleotides not longer than 29-mers. Here we show that ~150–400-mer model transcripts derived from [...] Read more.

Oligonucleotide conjugates of tris(2-aminobenzimidazole) have been reported previously to cleave complementary RNA strands with high levels of sequence and site specificity. The RNA substrates used in these studies were oligonucleotides not longer than 29-mers. Here we show that ~150–400-mer model transcripts derived from the 3′-untranslated region of the PIM1 mRNA reacted with rates and specificities comparable to those of short oligonucleotide substrates. The replacement of DNA by DNA/LNA mixmers further increased the cleavage rate. Tris(2-aminobenzimidazoles) were designed to interact with phosphates and phosphate esters. A cell, however, contains large amounts of phosphorylated species that may cause competitive inhibition of RNA cleavage. It is thus important to note that no loss in reaction rates was observed in phosphate buffer. This opens the way to in-cell applications for this type of artificial nuclease. Furthermore, we disclose a new synthetic method giving access to tris(2-aminobenzimidazoles) in multigram amounts. Full article

(This article belongs to the Special Issue Molecular Tools for Nucleic Acid Manipulation for Biological Intervention)

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12 pages, 1345 KiB

Open AccessArticle

Further Probing of Cu²⁺-Dependent PNAzymes Acting as Artificial RNA Restriction Enzymes

by Olivia Luige, Merita Murtola, Alice Ghidini and Roger Strömberg

Molecules 2019, 24(4), 672; https://doi.org/10.3390/molecules24040672 - 14 Feb 2019

Cited by 9 | Viewed by 4368

Abstract

Peptide nucleic acid (PNA)-neocuproine conjugates have been shown to efficiently catalyse the cleavage of RNA target sequences in the presence of Cu²⁺ ions in a site-specific manner. These artificial enzymes are designed to force the formation of a bulge in the RNA [...] Read more.

Peptide nucleic acid (PNA)-neocuproine conjugates have been shown to efficiently catalyse the cleavage of RNA target sequences in the presence of Cu²⁺ ions in a site-specific manner. These artificial enzymes are designed to force the formation of a bulge in the RNA target, the sequence of which has been shown to be key to the catalytic activity. Here, we present a further investigation into the action of Cu²⁺-dependent PNAzymes with respect to the dependence on bulge composition in 3- and 4-nucleotide bulge systems. Cu²⁺-dependent PNAzymes were shown to have a clear preference for 4-nucleotide bulges, as the cleavage of 3-nucleotide bulge-forming RNA sequences was significantly slower, which is illustrated by a shift in the half-lives from approximately 30 min to 24 h. Nonetheless, the nucleotide preferences at different positions in the bulge displayed similar trends in both systems. Moreover, the cleavage site was probed by introducing critical chemical modifications to one of the cleavage site nucleotides of the fastest cleaved 4-nucleotide RNA bulge. Namely, the exclusion of the exocyclic amine of the central adenine and the replacement of the 2′-hydroxyl nucleophile with 2′-H or 2′-OMe substituents in the RNA severely diminished the rate of RNA cleavage by the Cu²⁺-dependent PNAzyme, giving insight into the mechanism of cleavage. Moreover, the shorter recognition arm of the RNA/PNAzyme complex was modified by extending the PNAzyme by two additional nucleobases. The new PNAzyme was able to efficiently promote the cleavage of RNA when fully hybridised to a longer RNA target and even outperform the previous fastest PNAzyme. The improvement was demonstrated in cleavage studies with stoichiometric amounts of either PNAzyme present, and the extended PNAzyme was also shown to give turnover with a 10-fold excess of the RNA target. Full article

(This article belongs to the Special Issue Molecular Tools for Nucleic Acid Manipulation for Biological Intervention)

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13 pages, 2189 KiB

Open AccessArticle

Synthesis of Glycosidic (β-1′′→6, 3′ and 4′) Site Isomers of Neomycin B and Their Effect on RNA and DNA Triplex Stability

by Lotta Granqvist, Ville Tähtinen and Pasi Virta

Molecules 2019, 24(3), 580; https://doi.org/10.3390/molecules24030580 - 6 Feb 2019

Cited by 1 | Viewed by 2837

Abstract

Glycosidic (β-1′′→6, 3′ and 4′) site isomers of neomycin B (i.e., neobiosamine (β-1′′→6, 3′ and 4′) neamines) have been synthesized in a straightforward manner. Peracetylated neomycin azide was used as a common starting material to obtain neobiosamine glycosyl donor and 6, 3′,4′-tri-O [...] Read more.

Glycosidic (β-1′′→6, 3′ and 4′) site isomers of neomycin B (i.e., neobiosamine (β-1′′→6, 3′ and 4′) neamines) have been synthesized in a straightforward manner. Peracetylated neomycin azide was used as a common starting material to obtain neobiosamine glycosyl donor and 6, 3′,4′-tri-O-acetyl neamine azide that after simple protecting group manipulation was converted to three different glycosyl acceptors (i.e., 5,6,4′-, 5,3′,4′- and 5,6,3′-tri-O-acetyl neamine azide). Glycosylation between the neobiosamine glycosyl donor and the neamine-derived acceptors gave the protected pseudo-tetrasaccharides, which were converted, via global deprotection (deacetylation and reduction of the azide groups), to the desired site isomers of neomycin. The effect of these aminoglycosides on the RNA and DNA triplex stability was studied by UV-melting profile analysis. Full article

(This article belongs to the Special Issue Molecular Tools for Nucleic Acid Manipulation for Biological Intervention)

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17 pages, 2898 KiB

Open AccessArticle

Retro-1-Oligonucleotide Conjugates. Synthesis and Biological Evaluation

by Jordi Agramunt, Enrique Pedroso, Silvia M. Kreda, Rudolph L. Juliano and Anna Grandas

Molecules 2019, 24(3), 579; https://doi.org/10.3390/molecules24030579 - 6 Feb 2019

Cited by 4 | Viewed by 3841

Abstract

Addition of small molecule Retro-1 has been described to enhance antisense and splice switching oligonucleotides. With the aim of assessing the effect of covalently linking Retro-1 to the biologically active oligonucleotide, three different derivatives of Retro-1 were prepared that incorporated a phosphoramidite group, [...] Read more.

Addition of small molecule Retro-1 has been described to enhance antisense and splice switching oligonucleotides. With the aim of assessing the effect of covalently linking Retro-1 to the biologically active oligonucleotide, three different derivatives of Retro-1 were prepared that incorporated a phosphoramidite group, a thiol or a 1,3-diene, respectively. Retro-1–oligonucleotide conjugates were assembled both on-resin (coupling of the phosphoramidite) and from reactions in solution (Michael-type thiol-maleimide reaction and Diels-Alder cycloaddition). Splice switching assays with the resulting conjugates showed that they were active but that they provided little advantage over the unconjugated oligonucleotide in the well-known HeLa Luc705 reporter system. Full article

(This article belongs to the Special Issue Molecular Tools for Nucleic Acid Manipulation for Biological Intervention)

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11 pages, 2120 KiB

Open AccessArticle

Two-Holder Strategy for Efficient and Selective Synthesis of Lk 1 ssDNA Catenane

by Qi Li, Jing Li, Yixiao Cui, Sheng Liu, Ran An, Xingguo Liang and Makoto Komiyama

Molecules 2018, 23(9), 2270; https://doi.org/10.3390/molecules23092270 - 5 Sep 2018

Cited by 4 | Viewed by 3871

Abstract

DNA catenanes are characterized by their flexible and dynamic motions and have been regarded as one of the key players in sophisticated DNA-based molecular machines. There, the linking number (Lk) between adjacent interlocked rings is one of the most critical factors, since it [...] Read more.

DNA catenanes are characterized by their flexible and dynamic motions and have been regarded as one of the key players in sophisticated DNA-based molecular machines. There, the linking number (Lk) between adjacent interlocked rings is one of the most critical factors, since it governs the feasibility of dynamic motions. However, there has been no established way to synthesize catenanes in which Lk is controlled to a predetermined value. This paper reports a new methodology to selectively synthesize Lk 1 catenanes composed of single-stranded DNA rings, in which these rings can most freely rotate each other due to minimal inter-ring interactions. To the mixture for the synthesis, two holder strands (oligonucleotides of 18–46 nt) were added, and the structure of the quasi-catenane intermediate was interlocked through Watson–Crick base pairings into a favorable conformation for Lk 1 catenation. The length of the complementary part between the two quasi-rings was kept at 10 bp or shorter. Under these steric constraints, two quasi-rings were cyclized with the use of T4 DNA ligase. By this simple procedure, the formation of undesired topoisomers (Lk ≥ 2) was almost completely inhibited, and Lk 1 catenane was selectively prepared in high yield up to 70 mole%. These Lk 1 catenanes have high potentials as dynamic parts for versatile DNA architectures. Full article

(This article belongs to the Special Issue Molecular Tools for Nucleic Acid Manipulation for Biological Intervention)

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