Oligonucleotide Conjugates Research Articles

Selective recognition of RNA G-quadruplex in vitro and in cells by L-aptamer-D-oligonucleotide conjugate.

RNA Guanine-quadruplexes (rG4s) are important nucleic acid structures that govern vital biological processes. Although numerous tools have been developed to target rG4s, few specific tools are capable of discerning individual rG4 of interest. Herein, we design and synthesize the first L-aptamer-antisense oligonucleotide (ASO) conjugate, L-Apt.4-1c-ASO15nt(APP), with a focus on recognizing the amyloid precursor protein (APP) rG4 region as an example. The L-aptamer module binds with the rG4 structure, whereas ASO hybridizes with flanking sequences. Together, these two modules enhance the precise recognition of APP rG4. We demonstrate that the L-Apt.4-1c-ASO15nt(APP) conjugate can interact with the APP rG4 region with sub-nanomolar binding affinity, and distinguish APP rG4 from other G4s and non-G4s in vitro and in cells. We also show that L-Apt.4-1c-ASO15nt(APP) can inhibit APP protein expression. Notably, we investigate the inhibitory mechanism of this newly developed tool, and reveal that it controls gene expression by hindering DHX36 protein from unraveling the rG4, as well as by promoting translational inhibition and RNase H-mediated mRNA knockdown activity. Our novel L-aptamer-ASO conjugate tool not only enables the specific recognition of rG4 region of interest, but also allows efficient gene control via targeting rG4-containing transcripts in cells.

Novel NTCP ligand dimeric bile acid conjugated with ASO reduce hepatitis B virus surface antigen in vivo

Hepatitis B virus (HBV) specifically infects hepatocytes and causes severe liver diseases. However, functional cure is rarely attainable by current treatment modalities. Anti-sense oligonucleotide (ASO), which targets pregenomic RNAs to reduce hepatitis B virus (HBV) antigen production and viral replication, has been studied as a novel treatment strategy for HBV cure and can be conjugated with N-acetylgalactosamine (GalNAc), thereby enhancing hepatocyte uptake via the asialoglycoprotein receptor (ASGPR). In comparison to GalNAc-ASO conjugation, clinical research indicates that unconjugated ASO is more effective in reducing hepatitis B virus surface antigen level. Recent studies have revealed that human sodium taurocholate co-transporting polypeptide (NTCP) is a functional cellular receptor for hepatitis B virus (HBV), and the bivalent bile acid structure may interact with multiple binding sites on NTCP, yielding much stronger interaction and substantially improved binding affinity. In this study, we synthesized NTCP ligand-antisense oligonucleotide (ASO) conjugation and evaluated the potential of antiviral therapy specifically reduction of HBV antigenemia in mice in vivo.

Synthesis of Coumarin-oligonucleiotide Conjugates for Application in DNA-encoded Libraries

Oligonucleotides, including DNA and RNA, can be functionalized by chemical modification based on synthetic organic chemistry. For example, ligand-oligonucleotide conjugates have a wide variety of applications. Conjugates of functional ligands and oligonucleotides have attracted attention in recent years as a drug delivery system (DDS) for improving the efficacy of oligonucleotide therapeutics. In addition, oligonucleotide conjugates with drug candidate compounds as ligands have been applied to drug screening using DNA-encoded libraries (DELs). Against this background, we have focused on the development of practical synthetic methods for ligand-oligonucleotide conjugates. Recently, we have developed a new synthetic method to construct oligonucleotides conjugated with coumarins and dipeptides, which are expected to have bioactivity, for application to DDS research of oligonucleotide therapeutics and drug discovery research using DEL. In this review, we will discuss the details, including how to construct a coumarin scaffold on oligonucleotides based on Knoevenagel condensation.

Peptide-Oligonucleotide Conjugation: Chemistry and Therapeutic Applications.

Oligonucleotides have been identified as powerful therapeutics for treating genetic disorders and diseases related to epigenetic factors such as metabolic and immunological dysfunctions. However, they face certain obstacles in terms of limited delivery to tissues and poor cellular uptake due to their large size and often highly charged nature. Peptide-oligonucleotide conjugation is an extensively utilized approach for addressing the challenges associated with oligonucleotide-based therapeutics by improving their delivery, cellular uptake and bioavailability, consequently enhancing their overall therapeutic efficiency. In this review, we present an overview of the conjugation of oligonucleotides to peptides, covering the different strategies associated with the synthesis of peptide-oligonucleotide conjugates (POC), the commonly used peptides employed to generate POCs, with the aim to develop oligonucleotides with favourable pharmacokinetic (PK) or pharmacodynamic (PD) properties for therapeutic applications. The advantages and drawbacks of the synthetic methods and applications of POCs are also described.

Thiol-Specific Linkers for the Synthesis of Oligonucleotide Conjugates via Metal-Free Thiol-Ene Click Reaction.

Chemical conjugation of oligonucleotides is widely used to improve their delivery and therapeutic potential. A variety of strategies are implemented to efficiently modify oligonucleotides with conjugating partners. The linkers typically used for oligonucleotide conjugation have limitations in terms of stability or ease of synthesis, which generates the need for providing new improved linkers for oligonucleotide conjugation. Herein, we report the synthesis of novel vinylpyrimidine phosphoramidite building blocks, which can be incorporated into an oligonucleotide by standard solid-phase synthesis in an automated synthesizer. These linker-bearing oligonucleotides can be easily conjugated in a biocompatible manner with thiol-functionalized molecules leading to the efficient generation of oligonucleotide conjugates.

Targeting the Skin: The Study of a Bottlebrush Polymer-Antisense Oligonucleotide Conjugate in a Psoriasis Mouse Model.

The investigation of gene regulation therapeutics for the treatment of skin-related diseases is rarely explored in part due to inefficient systemic delivery. In this study, a bottlebrush polymer-antisense oligonucleotide (ASO) conjugate, termed pacDNA, designed to target IL-17 receptor A (IL-17RA), which is involved in psoriasis pathogenesis is presented. Systemic administration of pacDNA led to its accumulation in epidermis, dermis, and hypodermis of mouse skin, reduced IL-17RA gene expression in skin, and significantly reversed the development of imiquimod (IMQ)-induced psoriasis in a mouse model. These findings highlight the potential of the pacDNA as a promising nanoconstruct for systemic oligonucleotide delivery to the skin and for treating psoriasis and other skin-related disorders through systemic administration.

In vitro screening of chemically synthesized dipeptide-antisense oligonucleotide conjugates to identify ligand molecules enhancing their activity

Oligonucleotide therapeutics, particularly antisense oligonucleotides (ASOs), have emerged as promising candidates in drug discovery. However, their effective delivery to the target tissues and cells remains a challenge, necessitating the development of suitable drug delivery technologies for ASOs to enable their practical application. In this study, we synthesized a library of chemically modified dipeptide-ASO conjugates using a recent synthetic method based on the Ugi reaction. We then conducted in vitro screening of this library using luciferase-expressing cell lines to identify ligands capable of enhancing ASO activity. Our findings suggest that N-(4-nitrophenoxycarbonyl)glycine may interact with the thiophosphate moiety of the phosphorothioate-modification in ASO. Through our screening efforts, we identified two ligands that modestly reduced luciferase luminescence in a cell type-selective manner. Furthermore, quantification of luciferase mRNA levels revealed that one of these promising dipeptide-ASO conjugates markedly suppressed luciferase RNA levels through its antisense effect in prostate-derived DU-145 cells compared to the ASOs without ligand modification.

Incorporation of Intracellular NanoSIMS Tracers to Oligonucleotide Conjugates via Strain Promoted Sydnone-Alkyne Cycloaddition.

Extensive efforts have been dedicated to developing cell-specific targeting ligands that can be conjugated to therapeutic cargo, offering a promising yet still challenging strategy to deliver oligonucleotide therapeutics beyond the liver. Indeed, while the cargo and the ligand are crucial, the third component, the linker, is integral but is often overlooked. Here, we present strain-promoted sydnone-alkyne cycloaddition as a versatile linker chemistry for oligonucleotide synthesis, expanding the choices for bioconjugation of therapeutics while enabling subcellular detection of the linker and payload using nanoscale secondary ion mass spectrometry (NanoSIMS) imaging. This strategy was successfully applied to peptide and lipid ligands and profiled using the well characterized N-acetylgalactosamine (GalNAc) targeting ligand. The linker did not affect the expected activity of the conjugate and was detectable and distinguishable from the labeled cargo. Finally, this work not only offers a practical bioconjugation method but also enables the assessment of the linker's subcellular behavior, facilitating NanoSIMS imaging to monitor the three key components of therapeutic conjugates.

Phthalimide and 3‐Formylindole Derivatives for Oligonucleotide Conjugation by Amide or Oxime Ligation

A phthalimide or 3‐formylindole functionality was incorporated into a tris(hydroxymethyl)ethane (THME) scaffold, and the resulting compounds were subsequently transformed into phosphoramidites and solid supports These building blocks were then used to synthesise oligonucleotides containing amine or aldehyde groups at various positions within their sequences. The resulting modified oligonucleotides were further conjugated through either amide bond formation or oxime ligation. These novel building blocks expand the repertoire of phosphoramidites and solid supports available for modifying oligonucleotides.

Aptameric hirudins as selective and reversible EXosite-ACTive site (EXACT) inhibitors

Potent and selective inhibition of the structurally homologous proteases of coagulation poses challenges for drug development. Hematophagous organisms frequently accomplish this by fashioning peptide inhibitors combining exosite and active site binding motifs. Inspired by this biological strategy, we create several EXACT inhibitors targeting thrombin and factor Xa de novo by linking EXosite-binding aptamers with small molecule ACTive site inhibitors. The aptamer component within the EXACT inhibitor (1) synergizes with and enhances the potency of small-molecule active site inhibitors by many hundred-fold (2) can redirect an active site inhibitor’s selectivity towards a different protease, and (3) enable efficient reversal of inhibition by an antidote that disrupts bivalent binding. One EXACT inhibitor, HD22-7A-DAB, demonstrates extraordinary anticoagulation activity, exhibiting great potential as a potent, rapid onset anticoagulant to support cardiovascular surgeries. Using this generalizable molecular engineering strategy, selective, potent, and rapidly reversible EXACT inhibitors can be created against many enzymes through simple oligonucleotide conjugation for numerous research and therapeutic applications.

Molecular Pixelation: Spatial proteomics of single-cells by next generation sequencing

Abstract Molecular Pixelation (MPX) is a novel single cell spatial proteomics panel able to simultaneously quantify protein abundance, the spatial distribution, and colocalization of targeted cell surface proteins on up to 1,000 individual cells. MPX creates 3D spatial maps of cells by imprinting spatial information on antibody oligonucleotide conjugates using a DNA reagent we call DNA-pixels. These DNA-pixels form over 1,000 connected spatial zones per single cell producing graphs that reconstruct the cell surface in silico; forming a single cell surface map of 80 proteins. By applying spatial statistics on these cell surface graph representations, we uncover both known and novel patterns of protein spatial polarization and co-localization associated with vital immune processes such as intercellular communication and mobility. In our recent paper we show how MPX can be used to monitor the cell surface effects of treatment or stimulation, such as the spatial restructuring of the T cell surface proteins upon the formation of the uropod, the hind part of a migrating T cell. With high throughput and multiplex, MPX is a powerful tool for investigating the intricate spatial organization of proteins in single cells, paving the way for new discoveries in immune system research.

Freeze-Driven Synthesis of DNA Hairpin-Conjugated Gold Nanoparticle Biosensors for Dual-Mode Detection.

Freeze-based immobilization of deoxyribonucleic acid (DNA) oligonucleotides on gold nanoparticles (AuNPs) is highly efficient for single-stranded oligonucleotides but typically does not accommodate structures such as snap-cooled DNA hairpins (Sc-HPs) and snap-cooled molecular beacons (Sc-MBs) frequently used for biorecognition applications. Recognizing this limitation, we have developed a modified, freeze-based technique specifically designed to enable the adsorption of such hairpin oligonucleotides onto AuNP surfaces while ensuring that they retain their biosensing capabilities. Successful hairpin oligonucleotide conjugation of varying lengths to a wide range of AuNP diameters was corroborated by dynamic light scattering, ζ-potential, and UV-vis spectrophotometry. Moreover, we conducted a thorough evaluation of this modified method, confirming the retention of the sensing functions of Sc-HPs and Sc-MBs. This advancement not only offers a more efficient route for DNA hairpin conjugation but also elucidates the underlying biorecognition functions, with implications for broader applications in molecular diagnostics.

Quantifying the activity profile of ASO and siRNA conjugates in glioblastoma xenograft tumors in vivo.

Glioblastoma multiforme is a universally lethal brain tumor that largely resists current surgical and drug interventions. Despite important advancements in understanding GBM biology, the invasiveness and heterogeneity of these tumors has made it challenging to develop effective therapies. Therapeutic oligonucleotides-antisense oligonucleotides and small-interfering RNAs-are chemically modified nucleic acids that can silence gene expression in the brain. However, activity of these oligonucleotides in brain tumors remains inadequately characterized. In this study, we developed a quantitative method to differentiate oligonucleotide-induced gene silencing in orthotopic GBM xenografts from gene silencing in normal brain tissue, and used this method to test the differential silencing activity of a chemically diverse panel of oligonucleotides. We show that oligonucleotides chemically optimized for pharmacological activity in normal brain tissue do not show consistent activity in GBM xenografts. We then survey multiple advanced oligonucleotide chemistries for their activity in GBM xenografts. Attaching lipid conjugates to oligonucleotides improves silencing in GBM cells across several different lipid classes. Highly hydrophobic lipid conjugates cholesterol and docosanoic acid enhance silencing but at the cost of higher neurotoxicity. Moderately hydrophobic, unsaturated fatty acid and amphiphilic lipid conjugates still improve activity without compromising safety. These oligonucleotide conjugates show promise for treating glioblastoma.

The chemistry and biology of oligonucleotide conjugation

The chemistry and biology of oligonucleotide conjugation

Peptide‐Directed Attachment of Hydroxylamines to Specific Lysines of IgG Antibodies for Bioconjugations with Acylboronates

AbstractThe role of monoclonal antibodies as vehicles to deliver payloads has evolved as a powerful tool in cancer therapy in recent years. The clinical development of therapeutic antibody conjugates with precise payloads holds great promise for targeted therapeutic interventions. The use of affinity‐peptide mediated functionalization of native off‐the‐shelf antibodies offers an effective approach to selectively modify IgG antibodies with a drug–antibody ratio (DAR) of 2. Here, we report the traceless, peptide‐directed attachment of two hydroxylamines to native IgGs followed by chemoselective potassium acyltrifluoroborate (KAT) ligation with quinolinium acyltrifluoroborates (QATs), which provide enhanced ligation rates with hydroxylamines under physiological conditions. By applying KAT ligation to the modified antibodies, conjugation of small molecules, proteins, and oligonucleotides to off‐the‐shelf IgGs proceeds efficiently, in good yields, and with simultaneous cleavage of the affinity peptide‐directing moiety.

Peptide-Directed Attachment of Hydroxylamines to Specific Lysines of IgG Antibodies for Bioconjugations with Acylboronates.

The role of monoclonal antibodies as vehicles to deliver payloads has evolved as a powerful tool in cancer therapy in recent years. The clinical development of therapeutic antibody conjugates with precise payloads holds great promise for targeted therapeutic interventions. The use of affinity-peptide mediated functionalization of native off-the-shelf antibodies offers an effective approach to selectively modify IgG antibodies with a drug-antibody ratio (DAR) of 2. Here, we report the traceless, peptide-directed attachment of two hydroxylamines to native IgGs followed by chemoselective potassium acyltrifluoroborate (KAT) ligation with quinolinium acyltrifluoroborates (QATs), which provide enhanced ligation rates with hydroxylamines under physiological conditions. By applying KAT ligation to the modified antibodies, conjugation of small molecules, proteins, and oligonucleotides to off-the-shelf IgGs proceeds efficiently, in good yields, and with simultaneous cleavage of the affinity peptide-directing moiety.

A Convenient Oligonucleotide Conjugation via Tandem Staudinger Reaction and Amide Bond Formation at the Internucleotidic Phosphate Position.

Staudinger reaction on the solid phase between an electronodeficit organic azide, such as sulfonyl azide, and the phosphite triester formed upon phosphoramidite coupling is a convenient method for the chemical modification of oligonucleotides at the internucleotidic phosphate position. In this work, 4-carboxybenzenesulfonyl azide, either with a free carboxy group or in the form of an activated ester such as pentafluorophenyl, 4-nitrophenyl, or pentafluorobenzyl, was used to introduce a carboxylic acid function to the terminal or internal internucleotidic phosphate of an oligonucleotide via the Staudinger reaction. A subsequent treatment with excess primary alkyl amine followed by the usual work-up, after prior activation with a suitable peptide coupling agent such as a uronium salt/1-hydroxybenzotriazole in the case of a free carboxyl, afforded amide-linked oligonucleotide conjugates in good yields including multiple conjugations of up to the exhaustive modification at each phosphate position for a weakly activated pentafluorobenzyl ester, whereas more strongly activated and, thus, more reactive aryl esters provided only single conjugations at the 5'-end. The conjugates synthesized include those with di- and polyamines that introduce a positively charged side chain to potentially assist the intracellular delivery of the oligonucleotide.

Modular Synthesis of Methyl-Substituted Novel Psoralen N-Hydroxysuccinimide Esters and Evaluation of DNA Photocrosslinking Properties of the Corresponding Triplex-Forming Oligonucleotide Conjugates

AbstractPsoralen-conjugated triplex-forming oligonucleotides (Ps-TFOs) have been used to induce DNA mutations or to suppress gene expression through the formation of crosslinked products with DNA in a sequence-specific manner. Psoralen can crosslink with DNA at its furan-ring and/or pyrone-ring side, yielding either a monoadduct or diadduct (interstrand crosslinking) product. The differences in the crosslinked structures of Ps-TFOs with the target DNAs are closely related to the changes in the biological outcomes induced by the Ps-TFOs. However, only a few reports have discussed the photocrosslinking properties of Ps-TFOs. The photocrosslinking properties of Ps-TFOs with structurally diverse psoralen derivatives remain elusive. Herein, we report the modular synthesis of novel methyl-substituted psoralen N-hydroxysuccinimide (NHS) esters. By using these esters, the effect of the methyl substituent of psoralen on the photocrosslinking of the corresponding Ps-TFOs was examined. The amount of the diadduct product was significantly reduced in the presence of methyl substituents at the C-3 and C-4 positions, while the total amount of photocrosslinking product was maintained. This work demonstrates the possibility of controlling the crosslinked product of Ps-TFOs by introducing methyl groups into psoralen: this ability to manipulate the product is an important factor in the biological applications of Ps-TFOs.

Recent Updates of the CRISPR/Cas9 Genome Editing System: Novel Approaches to Regulate Its Spatiotemporal Control by Genetic and Physicochemical Strategies.

The genome editing approach by clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (CRISPR/Cas9) is a revolutionary advancement in genetic engineering. Owing to its simple design and powerful genome-editing capability, it offers a promising strategy for the treatment of different infectious, metabolic, and genetic diseases. The crystal structure of Streptococcus pyogenes Cas9 (SpCas9) in complex with sgRNA and its target DNA at 2.5 Å resolution reveals a groove accommodating sgRNA:DNA heteroduplex within a bilobate architecture with target recognition (REC) and nuclease (NUC) domains. The presence of a PAM is significantly required for target recognition, R-loop formation, and strand scission. Recently, the spatiotemporal control of CRISPR/Cas9 genome editing has been considerably improved by genetic, chemical, and physical regulatory strategies. The use of genetic modifiers anti-CRISPR proteins, cell-specific promoters, and histone acetyl transferases has uplifted the application of CRISPR/Cas9 as a future-generation genome editing tool. In addition, interventions by chemical control, small-molecule activators, oligonucleotide conjugates and bioresponsive delivery carriers have improved its application in other areas of biological fields. Furthermore, the intermediation of physical control by using heat-, light-, magnetism-, and ultrasound-responsive elements attached to this molecular tool has revolutionized genome editing further. These strategies significantly reduce CRISPR/Cas9's undesirable off-target effects. However, other undesirable effects still offer some challenges for comprehensive clinical translation using this genome-editing approach. In this review, we summarize recent advances in CRISPR/Cas9 structure, mechanistic action, and the role of small-molecule activators, inhibitors, promoters, and physical approaches. Finally, off-target measurement approaches, challenges, future prospects, and clinical applications are discussed.

Complexes and supramolecular associates of dodecyl-containing oligonucleotides with serum albumin

Serum albumin is currently in the focus of biomedical research as a promising platform for the creation of multicomponent self-assembling systems due to the presence of several sites with a high binding affinity of various compounds in its molecule, including lipophilic oligonucleotide conjugates. In this work, we investigated the stoichiometry of the dodecyl-containing oligonucleotides binding to bovine and human serum albumins via electrophoretic mobility shift assay. The results testify to the formation of albumin-oligonucleotide complexes with a stoichiometry of about 1 : (1.25 ± 0.25) under physiological-like conditions. Using atomic force microscopy, it was found that interaction of human serum albumin with a duplex of complementary dodecyl-containing oligonucleotides resulted in associates of round shape with a diameter of 165.5 ± 94.3 nm and 28.9 ± 16.9 nm in height, and with polydeoxyadenylic acid and dodecyl-containing oligothymidylate also resulted in formation of supramolecular associates with a size of about 315.4 ± 70.9 and 188.3 ± 43.7 nm. The obtained data allow considering dodecyl-containing oligonucleotides and albumin as potential components for the development of methods to design self-assembled systems for solving problems of molecular biology, biomedicine, and the development of unique theranostics with targeted action.