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15 pages, 2249 KiB

Open AccessArticle

A Versatile Solid-Phase Approach to the Synthesis of Oligonucleotide Conjugates with Biodegradable Hydrazone Linker

by Mariya I. Meschaninova, Nina S. Entelis, Elena L. Chernolovskaya and Alya G. Venyaminova

Molecules 2021, 26(8), 2119; https://doi.org/10.3390/molecules26082119 - 7 Apr 2021

Cited by 5 | Viewed by 3386

Abstract

One of the ways to efficiently deliver various drugs, including therapeutic nucleic acids, into the cells is conjugating them with different transport ligands via labile or stable bonds. A convenient solid-phase approach for the synthesis of 5′-conjugates of oligonucleotides with biodegradable pH-sensitive hydrazone [...] Read more.

One of the ways to efficiently deliver various drugs, including therapeutic nucleic acids, into the cells is conjugating them with different transport ligands via labile or stable bonds. A convenient solid-phase approach for the synthesis of 5′-conjugates of oligonucleotides with biodegradable pH-sensitive hydrazone covalent bonds is proposed in this article. The approach relies on introducing a hydrazide of the ligand under aqueous/organic media to a fully protected support-bound oligonucleotide containing aldehyde function at the 5′-end. We demonstrated the proof-of-principle of this approach by synthesizing 5′-lipophilic (e.g., cholesterol and α-tocopherol) conjugates of modified siRNA and non-coding RNAs imported into mitochondria (antireplicative RNAs and guide RNAs for Mito-CRISPR/system). The developed method has the potential to be extended for the synthesis of pH-sensitive conjugates of oligonucleotides of different types (ribo-, deoxyribo-, 2′-O-methylribo-, and others) with ligands of different nature. Full article

(This article belongs to the Special Issue Synthesis and Applications of Oligonucleotide Conjugate II)

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11 pages, 1686 KiB

Open AccessCommunication

Expanding the Scope of the Cleavable N-(Methoxy)oxazolidine Linker for the Synthesis of Oligonucleotide Conjugates

by Aapo Aho, Antti Äärelä, Heidi Korhonen and Pasi Virta

Molecules 2021, 26(2), 490; https://doi.org/10.3390/molecules26020490 - 18 Jan 2021

Cited by 4 | Viewed by 3254

Abstract

Oligonucleotides modified by a 2′-deoxy-2′-(N-methoxyamino) ribonucleotide react readily with aldehydes in slightly acidic conditions to yield the corresponding N-(methoxy)oxazolidine-linked oligonucleotide-conjugates. The reaction is reversible and dynamic in slightly acidic conditions, while the products are virtually stable above pH 7, where [...] Read more.

Oligonucleotides modified by a 2′-deoxy-2′-(N-methoxyamino) ribonucleotide react readily with aldehydes in slightly acidic conditions to yield the corresponding N-(methoxy)oxazolidine-linked oligonucleotide-conjugates. The reaction is reversible and dynamic in slightly acidic conditions, while the products are virtually stable above pH 7, where the reaction is in a ‘‘switched off-state’’. Small molecular examinations have demonstrated that aldehyde constituents affect the cleavage rate of the N-(methoxy)oxazolidine-linkage. This can be utilized to adjust the stability of this pH-responsive cleavable linker for drug delivery applications. In the present study, Fmoc-β-Ala-H was immobilized to a serine-modified ChemMatrix resin and used for the automated assembly of two peptidealdehydes and one aldehyde-modified peptide nucleic acid (PNA). In addition, a triantennary N-acetyl-d-galactosamine-cluster with a β-Ala-H unit has been synthesized. These aldehydes were conjugated via N-(methoxy)oxazolidine-linkage to therapeutically relevant oligonucleotide phosphorothioates and one DNA-aptamer in 19–47% isolated yields. The cleavage rates of the conjugates were studied in slightly acidic conditions. In addition to the diverse set of conjugates synthesized, these experiments and a comparison to published data demonstrate that the simple conversion of Gly-H to β-Ala-H residue resulted in a faster cleavage of the N-(methoxy)oxazolidine-linker at pH 5, being comparable (T_0.5 ca 7 h) to hydrazone-based structures. Full article

(This article belongs to the Special Issue Synthesis and Applications of Oligonucleotide Conjugate II)

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16 pages, 1788 KiB

Open AccessArticle

Synthesis of 5′-Thiamine-Capped RNA

by Marvin Möhler, Katharina Höfer and Andres Jäschke

Molecules 2020, 25(23), 5492; https://doi.org/10.3390/molecules25235492 - 24 Nov 2020

Cited by 9 | Viewed by 4662

Abstract

RNA 5′-modifications are known to extend the functional spectrum of ribonucleotides. In recent years, numerous non-canonical 5′-modifications, including adenosine-containing cofactors from the group of B vitamins, have been confirmed in all kingdoms of life. The structural component of thiamine adenosine triphosphate (thiamine-ATP), a [...] Read more.

RNA 5′-modifications are known to extend the functional spectrum of ribonucleotides. In recent years, numerous non-canonical 5′-modifications, including adenosine-containing cofactors from the group of B vitamins, have been confirmed in all kingdoms of life. The structural component of thiamine adenosine triphosphate (thiamine-ATP), a vitamin B1 derivative found to accumulate in Escherichia coli and other organisms in response to metabolic stress conditions, suggests an analogous function as a 5′-modification of RNA. Here, we report the synthesis of thiamine adenosine dinucleotides and the preparation of pure 5′-thiamine-capped RNAs based on phosphorimidazolide chemistry. Furthermore, we present the incorporation of thiamine-ATP and thiamine adenosine diphosphate (thiamine-ADP) as 5′-caps of RNA by T7 RNA polymerase. Transcripts containing the thiamine modification were modified specifically with biotin via a combination of thiazole ring opening, nucleophilic substitution and copper-catalyzed azide-alkyne cycloaddition. The highlighted methods provide easy access to 5′-thiamine RNA, which may be applied in the development of thiamine-specific RNA capture protocols as well as the discovery and confirmation of 5′-thiamine-capped RNAs in various organisms. Full article

(This article belongs to the Special Issue Synthesis and Applications of Oligonucleotide Conjugate II)

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18 pages, 4069 KiB

Open AccessArticle

Dependence of Fluorescence Quenching of CY3 Oligonucleotide Conjugates on the Oxidation Potential of the Stacking Base Pair

by Jens Sobek and Ralph Schlapbach

Molecules 2020, 25(22), 5369; https://doi.org/10.3390/molecules25225369 - 17 Nov 2020

Cited by 7 | Viewed by 3324

Abstract

To understand the complex fluorescence properties of astraphloxin (CY3)-labelled oligonucleotides, it is necessary to take into account the redox properties of the nucleobases. In oligonucleotide hybrids, we observed a dependence of the fluorescence intensity on the oxidation potential of the neighbouring base pair. [...] Read more.

To understand the complex fluorescence properties of astraphloxin (CY3)-labelled oligonucleotides, it is necessary to take into account the redox properties of the nucleobases. In oligonucleotide hybrids, we observed a dependence of the fluorescence intensity on the oxidation potential of the neighbouring base pair. For the series I < A < G < 8-oxoG, the extent of fluorescence quenching follows the trend of decreasing oxidation potentials. In a series of 7 nt hybrids, stacking interactions of CY3 with perfect match and mismatch base pairs were found to stabilise the hybrid by 7–8 kJ/mol. The fluorescence measurements can be explained by complex formation resulting in fluorescence quenching that prevails over the steric effect of a reduced excited state trans-cis isomerisation, which was expected to increase the fluorescence efficiency of the dye when stacking to a base pair. This can be explained by the fact that, in a double strand, base pairing and stacking cause a dramatic change in the oxidation potential of the nucleobases. In single-molecule fluorescence measurements, the oxidation of G to 8-oxoG was observed as a result of photoinduced electron transfer and subsequent chemical reactions. Our results demonstrate that covalently linked CY3 is a potent oxidant towards dsDNA. Sulfonated derivatives should be used instead. Full article

(This article belongs to the Special Issue Synthesis and Applications of Oligonucleotide Conjugate II)

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15 pages, 4572 KiB

Open AccessArticle

Highly Multiplexed Single-Cell In Situ RNA and DNA Analysis by Consecutive Hybridization

by Lu Xiao, Renjie Liao and Jia Guo

Molecules 2020, 25(21), 4900; https://doi.org/10.3390/molecules25214900 - 23 Oct 2020

Cited by 8 | Viewed by 5111

Abstract

The ability to comprehensively profile nucleic acids in individual cells in their natural spatial contexts is essential to advance our understanding of biology and medicine. Here, we report a novel method for spatial transcriptomics and genomics analysis. In this method, every nucleic acid [...] Read more.

The ability to comprehensively profile nucleic acids in individual cells in their natural spatial contexts is essential to advance our understanding of biology and medicine. Here, we report a novel method for spatial transcriptomics and genomics analysis. In this method, every nucleic acid molecule is detected as a fluorescent spot at its natural cellular location throughout the cycles of consecutive fluorescence in situ hybridization (C-FISH). In each C-FISH cycle, fluorescent oligonucleotide probes hybridize to the probes applied in the previous cycle, and also introduce the binding sites for the next cycle probes. With reiterative cycles of hybridization, imaging and photobleaching, the identities of the varied nucleic acids are determined by their unique color sequences. To demonstrate the feasibility of this method, we show that transcripts or genomic loci in single cells can be unambiguously quantified with 2 fluorophores and 16 C-FISH cycles or with 3 fluorophores and 9 C-FISH cycles. Without any error correction, the error rates obtained using the raw data are close to zero. These results indicate that C-FISH potentially enables tens of thousands (2¹⁶ = 65,536 or 3⁹ = 19,683) of different transcripts or genomic loci to be precisely profiled in individual cells in situ. Full article

(This article belongs to the Special Issue Synthesis and Applications of Oligonucleotide Conjugate II)

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12 pages, 1512 KiB

Open AccessArticle

A Trisbenzimidazole Phosphoramidite Building Block Enables High-Yielding Syntheses of RNA-Cleaving Oligonucleotide Conjugates

by Felix Zellmann and Michael W. Göbel

Molecules 2020, 25(8), 1842; https://doi.org/10.3390/molecules25081842 - 16 Apr 2020

Cited by 6 | Viewed by 3023

Abstract

The RNA cleaving catalyst tris(2-aminobenzimidazole) when attached to the 5’ terminus of oligonucleotides cuts complementary RNA strands in a highly site-specific manner. Conjugation was previously achieved by the acylation of an amino linker by an active ester of the catalyst. However, this procedure [...] Read more.

The RNA cleaving catalyst tris(2-aminobenzimidazole) when attached to the 5’ terminus of oligonucleotides cuts complementary RNA strands in a highly site-specific manner. Conjugation was previously achieved by the acylation of an amino linker by an active ester of the catalyst. However, this procedure was low yielding and not reliable. Here, a phosphoramidite building block is described that can be coupled to oligonucleotides by manual solid phase synthesis in total yields around 85%. Based on this chemistry, we have now studied the impact of LNA (locked nucleic acids) nucleotides on the rates and the site-specificities of RNA cleaving conjugates. The highest reaction rates and the most precise cuts can be expected when the catalyst is attached to a strong 5’ closing base pair and when the oligonucleotide contains several LNA units that are equally distributed in the strand. However, when placed in the 5’ position, LNA building blocks tend to diminish the specificity of RNA cleavage. Full article

(This article belongs to the Special Issue Synthesis and Applications of Oligonucleotide Conjugate II)

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Review

Jump to: Research

36 pages, 43812 KiB

Open AccessReview

Chemistry of Peptide-Oligonucleotide Conjugates: A Review

by Kristina Klabenkova, Alesya Fokina and Dmitry Stetsenko

Molecules 2021, 26(17), 5420; https://doi.org/10.3390/molecules26175420 - 6 Sep 2021

Cited by 52 | Viewed by 11851

Abstract

Peptide-oligonucleotide conjugates (POCs) represent one of the increasingly successful albeit costly approaches to increasing the cellular uptake, tissue delivery, bioavailability, and, thus, overall efficiency of therapeutic nucleic acids, such as, antisense oligonucleotides and small interfering RNAs. This review puts the subject of chemical [...] Read more.

Peptide-oligonucleotide conjugates (POCs) represent one of the increasingly successful albeit costly approaches to increasing the cellular uptake, tissue delivery, bioavailability, and, thus, overall efficiency of therapeutic nucleic acids, such as, antisense oligonucleotides and small interfering RNAs. This review puts the subject of chemical synthesis of POCs into the wider context of therapeutic oligonucleotides and the problem of nucleic acid drug delivery, cell-penetrating peptide structural types, the mechanisms of their intracellular transport, and the ways of application, which include the formation of non-covalent complexes with oligonucleotides (peptide additives) or covalent conjugation. The main strategies for the synthesis of POCs are viewed in detail, which are conceptually divided into (a) the stepwise solid-phase synthesis approach and (b) post-synthetic conjugation either in solution or on the solid phase, especially by means of various click chemistries. The relative advantages and disadvantages of both strategies are discussed and compared. Full article

(This article belongs to the Special Issue Synthesis and Applications of Oligonucleotide Conjugate II)

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27 pages, 1566 KiB

Open AccessReview

Site-Selective Artificial Ribonucleases: Renaissance of Oligonucleotide Conjugates for Irreversible Cleavage of RNA Sequences

by Yaroslav Staroseletz, Svetlana Gaponova, Olga Patutina, Elena Bichenkova, Bahareh Amirloo, Thomas Heyman, Daria Chiglintseva and Marina Zenkova

Molecules 2021, 26(6), 1732; https://doi.org/10.3390/molecules26061732 - 19 Mar 2021

Cited by 6 | Viewed by 4230

Abstract

RNA-targeting therapeutics require highly efficient sequence-specific devices capable of RNA irreversible degradation in vivo. The most developed methods of sequence-specific RNA cleavage, such as siRNA or antisense oligonucleotides (ASO), are currently based on recruitment of either intracellular multi-protein complexes or enzymes, leaving alternative [...] Read more.

RNA-targeting therapeutics require highly efficient sequence-specific devices capable of RNA irreversible degradation in vivo. The most developed methods of sequence-specific RNA cleavage, such as siRNA or antisense oligonucleotides (ASO), are currently based on recruitment of either intracellular multi-protein complexes or enzymes, leaving alternative approaches (e.g., ribozymes and DNAzymes) far behind. Recently, site-selective artificial ribonucleases combining the oligonucleotide recognition motifs (or their structural analogues) and catalytically active groups in a single molecular scaffold have been proven to be a great competitor to siRNA and ASO. Using the most efficient catalytic groups, utilising both metal ion-dependent (Cu(II)-2,9-dimethylphenanthroline) and metal ion-free (Tris(2-aminobenzimidazole)) on the one hand and PNA as an RNA recognising oligonucleotide on the other, allowed site-selective artificial RNases to be created with half-lives of 0.5–1 h. Artificial RNases based on the catalytic peptide [(ArgLeu)₂Gly]₂ were able to take progress a step further by demonstrating an ability to cleave miRNA-21 in tumour cells and provide a significant reduction of tumour growth in mice. Full article

(This article belongs to the Special Issue Synthesis and Applications of Oligonucleotide Conjugate II)

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14 pages, 1854 KiB

Open AccessReview

Conditionally Activated (“Caged”) Oligonucleotides

by Linlin Yang and Ivan J. Dmochowski

Molecules 2021, 26(5), 1481; https://doi.org/10.3390/molecules26051481 - 9 Mar 2021

Cited by 9 | Viewed by 4263

Abstract

Conditionally activated (“caged”) oligonucleotides provide useful spatiotemporal control for studying dynamic biological processes, e.g., regulating in vivo gene expression or probing specific oligonucleotide targets. This review summarizes recent advances in caging strategies, which involve different stimuli in the activation step. Oligo cyclization is [...] Read more.

Conditionally activated (“caged”) oligonucleotides provide useful spatiotemporal control for studying dynamic biological processes, e.g., regulating in vivo gene expression or probing specific oligonucleotide targets. This review summarizes recent advances in caging strategies, which involve different stimuli in the activation step. Oligo cyclization is a particularly attractive caging strategy, which simplifies the probe design and affords oligo stabilization. Our laboratory developed an efficient synthesis for circular caged oligos, and a circular caged antisense DNA oligo was successfully applied in gene regulation. A second technology is Transcriptome In Vivo Analysis (TIVA), where caged oligos enable mRNA isolation from single cells in living tissue. We highlight our development of TIVA probes with improved caging stability. Finally, we illustrate the first protease-activated oligo probe, which was designed for caspase-3. This expands the toolkit for investigating the transcriptome under a specific physiologic condition (e.g., apoptosis), particularly in specimens where light activation is impractical. Full article

(This article belongs to the Special Issue Synthesis and Applications of Oligonucleotide Conjugate II)

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