Modification Of Oligonucleotides Research Articles

Development of Phosphorothioate DNA and DNA Thioaptamers.

Nucleic acid aptamers are short RNA- or DNA-based affinity reagents typically selected from combinatorial libraries to bind to a specific target such as a protein, a small molecule, whole cells or even animals. Aptamers have utility in the development of diagnostic, imaging and therapeutic applications due to their size, physico-chemical nature and ease of synthesis and modification to suit the application. A variety of oligonucleotide modifications have been used to enhance the stability of aptamers from nuclease degradation in vivo. The non-bridging oxygen atoms of the phosphodiester backbones of RNA and DNA aptamers can be substituted with one or two sulfur atoms, resulting in thioaptamers with phosphorothioate or phosphorodithioate linkages, respectively. Such thioaptamers are known to have increased binding affinity towards their target, as well as enhanced resistance to nuclease degradation. In this review, we discuss the development of phosphorothioate chemistry and thioaptamers, with a brief review of selection methods.

Collagen Membranes with Ribonuclease Inhibitors for Long-Term Stability of Electrochemical Aptamer-Based Sensors Employing RNA.

Electrochemical aptamer-based (E-AB) sensors offer advantageous analytical detection abilities due to their rapid response time (seconds to minutes), specificity to a target, and selectivity to function in complex media. Ribonucleic acid (RNA) aptamers employed in this class of sensor offer favorable binding characteristics resulting from the ability of RNA to form stable tertiary folds aided by long-range intermolecular interactions. As a result, RNA aptamers can fold into three-dimensional structures more complex than those of their DNA counterparts and consequently exhibit better binding ability to target analytes. Unfortunately, RNA aptamers are susceptible to degradation by nucleases, and for this reason, RNA-based sensors are scarce or require significant sample pretreatment before use in clinically relevant media. Here, we combine the usefulness of a collagen I hydrogel membrane with entrapped ribonuclease inhibitors (RI) to protect small molecule RNA E-AB sensors from endogenous nucleases in complex media. More specifically, the biocompatibility of the naturally polymerized hydrogel with encapsulated RI promotes the protection of an aminoglycoside-binding RNA E-AB sensor up to 6 h, enabling full sensor function in nuclease-rich environments (undiluted serum) without the need for prior sample preparation or oligonucleotide modification. The use of collagen as a biocompatible membrane represents a general approach to compatibly interface E-AB sensors with complex biological samples.

Oligonucleotide modifications enhance probe stability for single cell transcriptome in vivo analysis (TIVA).

Single cell transcriptomics provides a powerful discovery tool for identifying new cell types and functions as well as a means to probe molecular features of the etiology and treatment of human diseases, including cancer. However, such analyses are limited by the difficulty of isolating mRNA from single cells within biological samples. We recently introduced a photochemical method for isolating mRNA from single living cells, Transcriptome In Vivo Analysis (TIVA). The TIVA probe is a "caged" polyU : polyA oligonucleotide hairpin designed to enter live tissue, where site-specific activation with 405 nm laser reveals the polyU-biotin strand to bind mRNA in a target cell, enabling subsequent mRNA isolation and sequencing. The TIVA method is well suited for analysis of living cells in resected tissue, but has not yet been applied to living cells in whole organisms. Adapting TIVA to this more challenging environment requires a probe with higher thermal stability, more robust caging, and greater nuclease resistance. In this paper we present modifications to the original TIVA probe with multiple aspects of enhanced stability. These newer probes utilize an extended 22mer polyU capture strand with two 9mer polyA blocking strands ("22/9/9") for higher thermal stability pre-photolysis and improved mRNA capture affinity post-photolysis. The "22/9/9 GC" probe features a terminal GC pair to reduce pre-photolysis interactions with mRNA by more than half. The "PS-22/9/9" probe features a phosphorothioated backbone, which extends serum stability from <1 h to at least 48 h, and also mediates uptake into cultured human fibroblasts.

Triazole- and Tetrazole-Bridged Nucleic Acids: Synthesis, Duplex Stability, Nuclease Resistance, and in Vitro and in Vivo Antisense Potency.

Antisense oligonucleotides are attractive therapeutic agents for several types of disease. One of the most promising modifications of antisense oligonucleotides is the introduction of bridged nucleic acids. As we report here, we designed novel bridged nucleic acids, triazole-bridged nucleic acid (TrNA), and tetrazole-bridged nucleic acid (TeNA), whose sugar conformations are restricted to N-type by heteroaromatic ring-bridged structures. We then successfully synthesized TrNA and TeNA and introduced these monomers into oligonucleotides. In UV-melting experiments, TrNA-modified oligonucleotides exhibited increased binding affinity toward complementary RNA and decreased binding affinity toward complementary DNA, although TeNA-modified oligonucleotides were decomposed under the annealing conditions. Enzymatic degradation experiments demonstrated that introduction of TrNA at the 3'-terminus rendered oligonucleotides resistant to nuclease digestion. Furthermore, we tested the silencing potencies of TrNA-modified antisense oligonucleotides using in vitro and in vivo assays. These experiments revealed that TrNA-modified antisense oligonucleotides induced potent downregulation of gene expression in liver. In addition, TrNA-modified antisense oligonucleotides showed a tendency for increased liver biodistribution. Taken together, our findings indicate that TrNA is a good candidate for practical application in antisense methodology.

Non-nucleotide Modification of Anti-miRNA Oligonucleotides.

MicroRNAs (miRNAs) are important modulators of gene expression. Synthetic anti-microRNA oligonucleotides (AMOs, or anti-miRs) are a form of steric-blocking antisense oligonucleotides (ASOs) that inhibit miRNA function through high-affinity binding and subsequent inactivation and/or degradation of the targeted miRNA. AMOs are a primary tool used to empirically determine the biological targets of a miRNA and can also be used therapeutically when overexpression of a miRNA contributes to a disease state. Chemical modification of synthetic AMOs enhance potency by protecting the oligonucleotide from nuclease degradation and by increasing binding affinity to the target miRNA. A new steric-blocking ASO modification strategy with favorable properties for use in AMOs was recently developed that combines use of high-affinity 2'-O-methyl RNA with terminally positioned non-nucleotide "ZEN" modifiers. This protocol describes use of ZEN AMOs in a dual-luciferase reporter assay as a simplified means to validate AMO performance or to quickly test putative miRNA binding sites in target sequences. This protocol also describes a method using Western blot analysis for quantifying the level of upregulation of proteins made from an mRNA that is thought to be under miRNA regulation, following inhibition of that miRNA by ZEN AMO treatment.

Antisense Oligonucleotides Internally Labeled with Peptides Show Improved Target Recognition and Stability to Enzymatic Degradation.

Specific target binding and stability in diverse biological media is of crucial importance for applications of synthetic oligonucleotides as diagnostic and therapeutic tools. So far, these issues have been addressed by chemical modification of oligonucleotides and by conjugation with a peptide, most often at the terminal position of the oligonucleotide. Herein, we for the first time systematically investigate the influence of internally attached short peptides on the properties of antisense oligonucleotides. We report the synthesis and internal double labeling of 21-mer oligonucleotides that target the BRAF V600E oncogene, with a library of rationally designed peptides employing CuAAC "click" chemistry. The peptide sequence has an influence on the specificity and affinity of target DNA/RNA binding. We also investigated the impact of locked nucleic acids (LNAs) on the latter. Lysine residues improve binding of POCs to target DNA and RNA, whereas the distance to lysine correlates exclusively with a decrease in binding of mismatched RNA targets. Glycine and tyrosine residues affect target binding as well. Importantly, the resistance of POCs to enzymatic degradation is dramatically improved by the internal attachment of peptides but not by LNA alone. Independently of the peptide sequence, the conjugates are stable for up to 24 h in 90% human serum and duplexes of POCs with complementary DNA for up to 160 h in 90% human serum. Such excellent stability has not been previously reported for DNA and makes internally labeled POCs an exciting object of study, i.e., showing high target specificity and simultaneous stability in biological media.

DNA functionalization by dynamic chemistry.

Dynamic combinatorial chemistry (DCC) is an attractive method to efficiently generate libraries of molecules from simpler building blocks by reversible reactions under thermodynamic control. Here we focus on the chemical modification of DNA oligonucleotides with acyclic diol linkers and demonstrate their potential for the deoxyribonucleic acid functionalization and generation of libraries of reversibly interconverting building blocks. The syntheses of phosphoramidite building blocks derived from D-threoninol are presented in two variants with protected amino or thiol groups. The threoninol building blocks were successfully incorporated via automated solid-phase synthesis into 13mer oligonucleotides. The amino group containing phosphoramidite was used together with complementary single-strand DNA templates that influenced the Watson–Crick base-pairing equilibrium in the mixture with a set of aldehyde modified nucleobases. A significant fraction of all possible base-pair mismatches was obtained, whereas, the highest selectivity (over 80%) was found for the guanine aldehyde templated by the complementary cytosine containing DNA. The elevated occurrence of mismatches can be explained by increased backbone plasticity derived from the linear threoninol building block as a cyclic deoxyribose analogue.

Development of a 3′-amino linker with high conjugation activity and its application to conveniently cross-link blunt ends of a duplex

The 2-aminoethyl carbamate linker (ssH linker) exhibits high activity in modifying the 5′-termini of oligonucleotides; however, the ssH linker is not appropriate for 3′-terminal modification because it undergoes intramolecular trans-acylation under heat–aqueous ammonia conditions. We developed an N-(2-aminoethyl)carbamate linker (revH linker), in which the carbamate is oriented in the reverse direction relative to that in 2-aminoethyl carbamate. The revH linker was tolerant to heat–alkaline conditions and retained its high reactivity in conjugation with exogenous molecules. The 3′-revH linker was efficiently linked with the 5′-ssH linker at the termini of complementary double strands with a bifunctional molecule, producing a synthetic loop structure. An anti-microRNA oligonucleotide (AMO) was prepared from the chemical ligation of three-stranded 2′-O-methyl RNAs, and the AMO with two alkyl loops exhibited high inhibition activity toward miRNA function. The revH linker is not only useful for 3′-terminal modification of oligonucleotides but also expands the utility range in combination with the 5′-ssH linker.

Targeting Insulin Receptor with a Novel Internalizing Aptamer

Nucleic acid-based aptamers are emerging as therapeutic antagonists of disease-associated proteins such as receptor tyrosine kinases. They are selected by an in vitro combinatorial chemistry approach, named Systematic Evolution of Ligands by Exponential enrichment (SELEX), and thanks to their small size and unique chemical characteristics, they possess several advantages over antibodies as diagnostics and therapeutics. In addition, aptamers that rapidly internalize into target cells hold as well great potential for their in vivo use as delivery tools of secondary therapeutic agents. Here, we describe a nuclease resistant RNA aptamer, named GL56, which specifically recognizes the insulin receptor (IR). Isolated by a cell-based SELEX method that allows enrichment for internalizing aptamers, GL56 rapidly internalizes into target cells and is able to discriminate IR from the highly homologous insulin-like growth factor receptor 1. Notably, when applied to IR expressing cancer cells, the aptamer inhibits IR dependent signaling. Given the growing interest in the insulin receptor as target for cancer treatment, GL56 reveals a novel molecule with great translational potential as inhibitor and delivery tool for IR-dependent cancers.

ChemInform Abstract: Palladium‐catalyzed Modification of Unprotected Nucleosides, Nucleotides, and Oligonucleotides

AbstractReview: 124 refs.

G-Quadruplex Forming Oligonucleotides as Anti-HIV Agents

Though a variety of different non-canonical nucleic acids conformations have been recognized, G-quadruplex structures are probably the structural motifs most commonly found within known oligonucleotide-based aptamers. This could be ascribed to several factors, as their large conformational diversity, marked responsiveness of their folding/unfolding processes to external stimuli, high structural compactness and chemo-enzymatic and thermodynamic stability. A number of G-quadruplex-forming oligonucleotides having relevant in vitro anti-HIV activity have been discovered in the last two decades through either SELEX or rational design approaches. Improved aptamers have been obtained by chemical modifications of natural oligonucleotides, as terminal conjugations with large hydrophobic groups, replacement of phosphodiester linkages with phosphorothioate bonds or other surrogates, insertion of base-modified monomers, etc. In turn, detailed structural studies have elucidated the peculiar architectures adopted by many G-quadruplex-based aptamers and provided insight into their mechanism of action. An overview of the state-of-the-art knowledge of the relevance of putative G-quadruplex forming sequences within the viral genome and of the most studied G-quadruplex-forming aptamers, selectively targeting HIV proteins, is here presented.

Palladium-catalyzed modification of unprotected nucleosides, nucleotides, and oligonucleotides.

Synthetic modification of nucleoside structures provides access to molecules of interest as pharmaceuticals, biochemical probes, and models to study diseases. Covalent modification of the purine and pyrimidine bases is an important strategy for the synthesis of these adducts. Palladium-catalyzed cross-coupling is a powerful method to attach groups to the base heterocycles through the formation of new carbon-carbon and carbon-heteroatom bonds. In this review, approaches to palladium-catalyzed modification of unprotected nucleosides, nucleotides, and oligonucleotides are reviewed. Polar reaction media, such as water or polar aprotic solvents, allow reactions to be performed directly on the hydrophilic nucleosides and nucleotides without the need to use protecting groups. Homogeneous aqueous-phase coupling reactions catalyzed by palladium complexes of water-soluble ligands provide a general approach to the synthesis of modified nucleosides, nucleotides, and oligonucleotides.

Cy3 and Cy5 dyes attached to oligonucleotide terminus stabilize DNA duplexes: Predictive thermodynamic model

Cyanine dyes are important chemical modifications of oligonucleotides exhibiting intensive and stable fluorescence at visible light wavelengths. When Cy3 or Cy5 dye is attached to 5′ end of a DNA duplex, the dye stacks on the terminal base pair and stabilizes the duplex. Using optical melting experiments, we have determined thermodynamic parameters that can predict the effects of the dyes on duplex stability quantitatively (ΔG°, Tm). Both Cy dyes enhance duplex formation by 1.2kcal/mol on average, however, this Gibbs energy contribution is sequence-dependent. If the Cy5 is attached to a pyrimidine nucleotide of pyrimidine–purine base pair, the stabilization is larger compared to the attachment to a purine nucleotide. This is likely due to increased stacking interactions of the dye to the purine of the complementary strand. Dangling (unpaired) nucleotides at duplex terminus are also known to enhance duplex stability. Stabilization originated from the Cy dyes is significantly larger than the stabilization due to the presence of dangling nucleotides. If both the dangling base and Cy3 are present, their thermodynamic contributions are approximately additive. New thermodynamic parameters improve predictions of duplex folding, which will help design oligonucleotide sequences for biophysical, biological, engineering, and nanotechnology applications.

プロドラッグ開発を目的とする合成核酸の保護と修飾

プロドラッグ開発を目的とする合成核酸の保護と修飾

High-throughput chemical modification of oligonucleotides for systematic structure-activity relationship evaluation.

Chemical modification of siRNA is achieved in a high-throughput manner (96-well plate format) by copper catalyzed azide-alkyne cycloadditions. This transformation can be performed in one synthetic operation at up to four positions with complete specificity, good yield, and acceptable purity. As demonstrated here, this approach extends the current synthetic options for oligonucleotide modifications and simultaneously facilitates the systematic, rapid biological evaluation of modified siRNA.

MiRNA-based therapies: strategies and delivery platforms for oligonucleotide and non-oligonucleotide agents.

The discovery of miRNAs as important regulatory agents for gene expression has expanded the therapeutic opportunities for oligonucleotides. In contrast to siRNA, miRNA-targeted therapy is able to influence not only a single gene, but entire cellular pathways or processes. It is possible to supplement downregulated or non-functional miRNAs by synthetic oligonucleotides, as well as alleviating effects caused by overexpression of malignant miRNAs through artificial antagonists, either oligonucleotides or small molecules. Chemical oligonucleotide modifications together with an efficient delivery system seem to be mandatory for successful therapeutic application. While miRNA-based therapy benefits from the decades of research spent on other therapeutic oligonucleotides, there are some specific challenges associated with miRNA therapy, mainly caused by the short target sequence. The current status and recent progress of miRNA-targeted therapeutics is described and future challenges and potential applications in treatment of cancer and viral infections are discussed.

163: Sequence-dependent off-target inhibition of TLR7/8 by synthetic microRNA inhibitors

Anti-microRNA (miRNA) oligonucleotides (AMOs) are commonly used to study miRNA function and present clear therapeutic potential. 2′-O-methyl (2′Ome) modified AMOs are among the most popular AMOs and can be modified to achieve high potency, high specificity and low cytoxicity. Nonetheless, 2′Ome oligonucleotide modification has previously been associated with an inhibitory activity on the sensing of pathogenic RNAs by innate immune sensors Toll-like receptor 7 and 8 (TLR7/8). In this work, we report on the ability of 2′Ome AMOs to inhibit TLR7 sensing of RNA in a sequence-dependent manner, independent of their miRNA-targeting function. Through a screen of 30 AMOs targeting common miRNAs, we found a subset of highly inhibitory sequences in mouse macrophages. Interspecies conservation of inhibitory activity on TLR7/8 was confirmed in human peripheral blood mononuclear cells – with an IC 50 activity as low as 2 nmol/l. Critically, we identified a core motif governing the inhibitory activity of these AMOs, which is present in more than 70 AMOs targeted to human miRNAs in miRBaseV20. DNA/LNA AMOs synthesised with a phosphorothioate backbone also inhibited TLR7 in a sequence-dependent manner, demonstrating that the off-target effects of AMOs on TLR7/8 are not restricted to 2′Ome AMOs. Collectively, our work establishes the potential for off-target effects of AMOs on TLR7/8 function, which should be taken into account when studying the role of miRNAs in immune responses.

Synthesis and properties of DNA oligonucleotides with a zwitterionic backbone structure.

The nucleosyl amino acid (NAA)-modification of oligonucleotides is introduced, which enables the preparation of oligonucleotides with zwitterionic backbone structures. It is demonstrated that partially zwitterionic NAA-modified DNA oligonucleotides are capable of duplex formation with native polyanionic counterstrands and show retained sensitivity towards base-pairing mismatches.

Photoactivated bioconjugation between ortho-azidophenols and anilines: a facile approach to biomolecular photopatterning.

Methods for the surface patterning of small molecules and biomolecules can yield useful platforms for drug screening, synthetic biology applications, diagnostics, and the immobilization of live cells. However, new techniques are needed to achieve the ease, feature sizes, reliability, and patterning speed necessary for widespread adoption. Herein, we report an easily accessible and operationally simple photoinitiated reaction that can achieve patterned bioconjugation in a highly chemoselective manner. The reaction involves the photolysis of 2-azidophenols to generate iminoquinone intermediates that couple rapidly to aniline groups. We demonstrate the broad functional group compatibility of this reaction for the modification of proteins, polymers, oligonucleotides, peptides, and small molecules. As a specific application, the reaction was adapted for the photolithographic patterning of azidophenol DNA on aniline glass substrates. The presence of the DNA was confirmed by the ability of the surface to capture living cells bearing the sequence complement on their cell walls or cytoplasmic membranes. Compared to other light-based DNA patterning methods, this reaction offers higher speed and does not require the use of a photoresist or other blocking material.

An Exceptionally Simple Strategy for DNA-Functionalized Up-Conversion Nanoparticles as Biocompatible Agents for Nanoassembly, DNA Delivery, and Imaging

Lanthanide-doped up-conversion nanoparticles (UCNPs) have shown promise in biomedical applications. However, as the UCNPs are normally capped with hydrophobic ligands, it remains challenging to prepare biocompatible UCNPs with specific molecular recognition capabilities. We herein report an exceptionally simple strategy to prepare uniform DNA-modified UCNPs as versatile bioprobes. The approach can directly convert as-prepared hydrophobic UCNPs into water-soluble DNA-UCNPs without any chemical modification of UCNPs or oligonucleotides. Furthermore, DNA molecules on the DNA-UCNPs retain their biorecognition ability, allowing programmable assembly of hybrid nanostructures. More importantly, we show that these DNA-UCNPs are capable of crossing cell membranes without the need of transfection agents, and their use as agents for bioimaging and DNA delivery are also demonstrated. Finally, DNA aptamer-conjugated UCNPs can be readily used for targeted imaging of cancer cells.