Search Results (210)

Modification of synthetic oligonucleotides and DNA is widely used in many applications in the life sciences. However, in most cases, modified DNA cannot be restored to its native state. Here, we report the preparation of a thymidine-inosine dimer building block (TID) for oligonucleotide synthesis. The TID modification supports the functionalization of synthetic oligonucleotides, which can later be removed to restore the DNA strand to its native state. The TID unit allows for a wide spectrum of postsynthetic modifications of oligonucleotides through click chemistry, including conjugation with fluorescent tags and small molecules, preparation of branched oligonucleotide scaffolds, and anchoring to a solid support. Due to the modification of the thymine base, the TID unit reduces the stability of the DNA duplex. We found that the negative effect of internal TID modification on duplex stability does not exceed the same for a single base mismatch. As long as the TID modification is present in the DNA strand, it disrupts its natural functionality. The “caging” effect of TID in the template strand with respect to DNA polymerase was demonstrated in primer extension experiments. Traceless removal of the temporary functional group occurs through oxidative cleavage of the inosine subunit, resulting in the formation of a native DNA strand with the thymine base left at the cleavage site. An anthracene-modified dodecamer oligonucleotide and a branched oligonucleotide scaffold were used to study the cleavage of the reporter group or the oligonucleotide side strand, respectively. It was shown that aqueous tetramethylguanidine efficiently cleaves the oxidized inosine subunit of TID at 37 °C, forming the native DNA strand. Full article

The WHO considers the Epidermal Growth Factor Receptor (EGFR) one of the key biomarkers of glioblastoma (GB). EGFR can be identified and targeted using molecular recognition elements (MoREs), like aptamers and aptamer–drug conjugates (ApDCs). Understanding the kinetics of anti-EGFR ApDC interactions with EGFR as well as the kinetics of their internalization into the cells is a crucial step for the further development of anti-EGFR ApDCs. For the first time, a novel approach was implemented to study real-time kinetics by measuring the cellular index (CI) using impedance (xCELLigence). Doxorubicin (DOX) was used as an indicator drug. Because DOX intercalates into the DNA double helix, aptamer–DOX non-covalent complexes were obtained. For the anti-EGFR DNA aptamer GR20, an additional duplex was constructed by synthesizing the extra region (GR20h) and via hybridization with the complementary oligonucleotide (h’) to form a duplex (hh’), thus creating the aptamer construct with complementary oligonucleotide (ACCO) GR20hh’. The original HPLC method quantified the assembly efficiency of an ACCO. The ACCO GR20hh’ retained affinity for the recombinant extracellular domain of EGFR, as measured using Biolayer Interferometry (BLI). According to cytofluorimetry, the ACCO GR20hh’ interacts with cells of continuous culture from GB patient (CCGBP) surgical samples. The DOX–ACCO GR20hh’ complexes are more efficiently internalized by EGFR+ cells lines A-431 and CCGBP 107 than DOX alone. Full article

Tumor immunotherapy has revolutionized cancer treatment by harnessing the immune system to recognize and eliminate malignant cells, with immune checkpoint inhibitors targeting programmed death receptor 1 (PD-1), programmed death-ligand 1 (PD-L1), and cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) demonstrating remarkable clinical success. However, challenges such as treatment resistance, immune-related adverse effects, and high costs highlight the need for novel therapeutic approaches. Aptamers, short, single-stranded oligonucleotides with high specificity and affinity for target molecules, have emerged as promising alternatives to conventional antibody-based therapies. This review provides a comprehensive analysis of aptamer-based strategies targeting immune checkpoints, with a particular focus on PD-1/PD-L1 and CTLA-4. We summarize recent advances in aptamer design, including bispecific and multifunctional aptamers, and explore their potential in overcoming immune resistance and improving therapeutic efficacy. Additionally, we discuss strategies to enhance aptamer stability, bioavailability, and tumor penetration through chemical modifications and nanoparticle conjugation. Preclinical and early clinical studies have demonstrated that aptamers can effectively block immune checkpoint pathways, restore T-cell activity, and synergize with other immunotherapeutic agents to achieve superior anti-tumor responses. By systematically reviewing the current research landscape and identifying key challenges, this review aims to provide valuable insights into the future directions of aptamer-based cancer immunotherapy, paving the way for more effective and personalized treatment strategies. Full article

Background/Objectives: Temozolomide is an important drug used for the treatment of glioblastoma multiforme. Covalent conjugation of temozolomide to triplex-forming oligonucleotides could facilitate better sequence discrimination when targeted to DNA to lessen off-target effects and potentially reduce side-effects associated with conventional chemotherapy. The base sensitivity of temozolomide precludes use of basic deprotection conditions that typify the solid-supported synthesis of oligonucleotides. Methods: A novel di-iso-propylsilylene-linked solid support was developed and used in solid-supported synthesis of oligonucleotide conjugates. Results: Conditions were established whereby fully deprotected, solid-supported oligonucleotides could be prepared for derivatisation. Cleavage of the di-iso-propylsilylene linker was possible using mild, acidic conditions. Conclusions: The di-iso-propylsilylene-linked solid support was developed and shown to be compatible with base-sensitive oligonucleotide conjugate formation. The DNA triplex formation exhibited by a temozolomide oligonucleotide conjugate was equal in stability to the unconjugated control, opening new possibilities for sequence selective delivery of temozolomide to targeted DNA. Full article

Lipoprotein (a) [Lp(a)] has been recognized as an independent, inherited, causal risk factor for atherosclerotic cardiovascular disease (ASCVD) and aortic valve stenosis, thus representing a major target of residual CV risk. Currently, no drug has been officially approved for lowering Lp(a) levels, and in clinical practice, Lp(a) is mainly used to (re)define CV risk, particularly in individuals at borderline CV risk and people with a family history of premature coronary heart disease, according to various guidelines. Specific Lp(a)-targeted antisense oligonucleotides (ASOs) and small interfering RNA (siRNA) agents have been developed to produce substantial Lp(a) reductions via the inhibition of apo(a) synthesis in the liver. These drugs are conjugated to N-acetylgalactosamine (GalNAc) to ensure their binding to asialoglycoproteins, which are specifically expressed on the surface of the hepatocytes. Such drugs include pelacarsen (an injectable ASO) and olpasiran, zerlasiran, and lepodisiran (injectable siRNA agents). Muvalaplin represents another therapeutic option to lower Lp(a) levels, since it is an oral selective small molecule inhibitor of Lp(a) formation, thus potentially exerting certain advantages in terms of its clinical use. The present narrative review summarizes the available clinical data on the efficacy and safety of these investigational Lp(a)-lowering therapies, as reported in phase 1 and 2 trials. The effects of these drugs on other [aside from Lp(a)] lipid parameters are also discussed. The phase 3 CV trial outcomes are ongoing for some of these agents (i.e., pelacarsen, olpasiran, and lepodisiran) and are briefly mentioned. Overall, there is an urgent need for evidence-based guidelines on Lp(a) reduction in daily clinical practice, following the results of the phase 3 CV trials, as well as for establishing the ideal Lp(a) quantification method (i.e., using an apo(a) isoform-independent assay with appropriate calibrators, reporting the Lp(a) level in molar units). Full article

The U.S. Food and Drug Administration (FDA) has authorized 50 new drugs in 2024, which matches the average figure for recent years (2018–2023). The approval of 13 monoclonal antibodies (mAbs) sets a new record, with these molecules accounting for more than 25% of all drugs authorized this year. Three proteins have been added to the list of biologics, and with the inclusion of four TIDES (two oligonucleotides and two peptides), only one in three approved drugs this year is a small molecule. As of 2023, no antibody-drug conjugates (ADCs) have reached the market this year. Two deuterated drugs have been approved, bringing the total approvals for this class of compounds to four. This year saw the authorization of two more PEGylated drugs—both peptides—highlighting a renewed interest in this strategy for extending drug half-life, despite the setback caused by the withdrawal of peginesatide from the market in 2014 due to adverse side effects. N-aromatic heterocycles and fluorine atoms are present in two-thirds of all the small molecules approved this year. Herein, the 50 new drugs authorized by the FDA in 2024 are analyzed exclusively on the basis of their chemical structure. They are classified as the following: biologics (antibodies, proteins), TIDES (oligonucleotides and peptides), combined drugs, natural products, F-containing molecules, nitrogen aromatic heterocycles, aromatic compounds, and other small molecules. Full article

Background: Systemic arterial hypertension contributes to cardiovascular morbidity and mortality worldwide. Many patients cannot achieve optimal blood pressure (BP) control with traditional therapies, which often results in poor patient adherence and limited long-term efficacy. We investigated the potential of RNA interference (RNAi) therapies targeting hepatic angiotensinogen (AGT) for hypertension management. Methods: This scoping review was conducted by the Joanna Briggs Institute, following a six-stage methodological framework and adhering to PRISMA recommendations. A comprehensive search was conducted across seven databases to identify relevant studies published until May 2024. Data extraction was performed separately, and both quantitative and qualitative analyses were conducted. A population, concept, and context model-based search was performed, selecting controlled MeSH terms and uncontrolled descriptors and cross-referencing them using Booleans. Results: Fifteen articles met our inclusion criteria. Focusing on the efficacy and safety of RNAi-based therapies, this review discusses several key approaches, including antisense oligonucleotides (IONIS-AGT-LRx), small interfering RNA (siRNAs; zilebesiran), and adeno-associated viruses carrying short hairpin RNAs. Notably, zilebesiran conjugated with N-acetylgalactosamine significantly reduced systolic BP by 20 mmHg, sustained for up to six months post-administration, with minimal adverse effects. Conclusions: RNAi-based therapies, particularly those using siRNAs, such as zilebesiran, are promising for the treatment of hypertension. They offer long-term BP control with fewer doses, potentially improving patient adherence and outcome. Although these therapies address several limitations of current antihypertensive treatments, further studies are required to confirm their long-term safety and efficacy. Full article

Therapeutic nucleic acids (TNAs) including antisense oligonucleotides (ASOs) and small interfering RNA (siRNA) have emerged as promising treatment strategies for a wide variety of diseases, offering the potential to modulate gene expression with a high degree of specificity. These small, synthetic nucleic acid-like molecules provide unique advantages over traditional pharmacological agents, including the ability to target previously “undruggable” genes. Despite this promise, several biological barriers severely limit their clinical efficacy. Upon administration, TNAs primarily enter cells through endocytosis, becoming trapped inside membrane-bound vesicles known as endosomes. Studies estimate that only 1–2% of TNAs successfully escape endosomal compartments to reach the cytosol, and in some cases the nucleus, where they bind target mRNA and exert their therapeutic effect. Endosomal entrapment and inefficient nuclear localization are therefore critical bottlenecks in the therapeutic application of TNAs. This review explores the current understanding of TNA endosomal escape and nuclear transport along with strategies aimed at overcoming these challenges, including the use of endosomal escape agents, peptide-TNA conjugates, non-viral delivery vehicles, and nuclear localization signals. By improving both endosomal escape and nuclear localization, significant advances in TNA-based therapeutics can be realized, ultimately expanding their clinical utility. Full article

N-Acetylgalactosamine (GalNAc) is an efficient and multifunctional delivery tool in the development and synthesis of chemically modified oligonucleotide therapeutics (conjugates). Such therapeutics demonstrate improved potency in vivo due to the selective and efficient delivery to hepatocytes in the liver via receptor-mediated endocytosis, which is what drives the high interest in this molecule. The ways to synthesize such structures are relatively new and have not been optimized in terms of the yields and stages both in lab and large-scale synthesis. Another significant criterion, especially in large-scale synthesis, is to match ecological norms and perform the synthesis in accordance with the Green Chemistry approach, i.e., to control and minimize the amounts of reagents and resources consumed and the waste generated. Here, we provide a robust and resource effective pot-economy method for the synthesis of triantennary GalNAc and GalNAc phosphoramidite/CPG optimized for laboratory scales. Full article

Telomerase presents over-expression in most cancer cells and has been used as a near-universal marker of cancer. Studies have revealed that inhibiting telomerase activity by utilizing oligonucleotides to down-regulate the expression of intracellular human telomerase reverse-transcriptase (hTERT) mRNA is an effective method of achieving anti-tumor therapy. Considering that oncogenic microRNA-21 has been proven to indirectly up-regulate hTERT expression and drive cancer metastasis and aggression through increased telomerase activity, here, we constructed an AS1411-functionallized oligonucleotide-conjugated gold nanoprobe (Au nanoprobe) to simultaneously down-regulate intracellular microRNA-21 and hTERT mRNA by using anti-sense oligonucleotide technology to explore their targeted anti-tumor therapy effect. In vitro cell studies demonstrated that Au nanoprobes could effectively induce apoptosis and inhibit the proliferation of cancer cells by down-regulating intracellular hTERT activity. In vivo imaging and anti-tumor studies revealed that Au nanoprobes could accumulate at the tumor site and inhibit the growth of MCF-7 tumor xenografted on balb/c nude mice, thus having potential for anti-tumor therapy. Full article

Recent advances in genetics and nucleic acid chemistry have created fundamentally new tools, both for practical applications in therapy and diagnostics and for fundamental genome editing tasks. Nucleic acid-based therapeutic agents offer a distinct advantage of selectively targeting the underlying cause of the disease. Nevertheless, despite the success achieved thus far, there remain unresolved issues regarding the improvement of the pharmacokinetic properties of therapeutic nucleic acids while preserving their biological activity. In order to address these challenges, there is a growing focus on the study of safe and effective delivery methods utilising modified nucleic acid analogues and their lipid bioconjugates. The present review article provides an overview of the current state of the art in the use of chemically modified nucleic acid derivatives for therapeutic applications, with a particular focus on oligonucleotides conjugated to lipid moieties. A systematic analysis has been conducted to investigate the ability of amphiphilic oligonucleotides to self-assemble into micelle-like structures, as well as the influence of non-covalent interactions of such derivatives with serum albumin on their biodistribution and therapeutic effects. Full article

The development of novel tools to tackle viral processes has become a central focus in global health, during the COVID-19 pandemic. The spike protein is currently one of the main SARS-CoV-2 targets, owing to its key roles in infectivity and virion formation. In this context, exploring innovative strategies to block the activity of essential factors of SARS-CoV-2, such as spike proteins, will strengthen the capacity to respond to current and future threats. In the present work, we developed and tested novel bispecific molecules that encompass: (i) oligonucleotide aptamers S901 and S702, which bind to the spike protein through its S1 domain, and (ii) hydrophobic tags, such as adamantane and tert-butyl-carbamate-based ligands. Hydrophobic tags have the capacity to trigger the degradation of targets recruited in the context of a proteolytic chimera by activating quality control pathways. We observed that S901-adamantyl conjugates promote the degradation of the S1 spike domain, stably expressed in human cells by genomic insertion. These results highlight the suitability of aptamers as target-recognition molecules and the robustness of protein quality control pathways triggered by hydrophobic signals, and place aptamer-Hytacs as promising tools for counteracting coronavirus progression in human cells. Full article

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. Full article

A promising and sought-after class of nanozymes for various applications is Pt-containing nanozymes, primarily Au@Pt, due to their easy preparation and remarkable catalytic properties. This study aimed to explore the freeze–thaw method for functionalizing Pt-containing nanozymes with oligonucleotides featuring a polyadenine anchor. Spherical gold nanoparticles ([Au]NPs) were synthesized and subsequently used as seeds to produce urchin-like Au@Pt nanoparticles ([Au@Pt]NPs) with an average diameter of 29.8 nm. The nanoparticles were conjugated with a series of non-thiolated DNA oligonucleotides, each consisting of three parts: a 5′-polyadenine anchor (A_n, with n = 3, 5, 7, 10; triple-branched A₃, or triple-branched A₅), a random sequence of 23 nucleotides, and a linear polyT block consisting of seven deoxythymine residues. The resulting conjugates were characterized using transmission electron microscopy, spectroscopy, dynamic light scattering, and emission detection of the fluorescent label at the 3′-end of each oligonucleotide. The stability of the conjugates was found to depend on the type of oligonucleotide, with decreased stability in the row of [Au@Pt]NP conjugates with A₇ > A₅ > 3A₃ > 3A₅ > A₁₀ > A₃ anchors. These [Au@Pt]NP–oligonucleotide conjugates were further evaluated using lateral flow test strips to assess fluorescein-specific binding and peroxidase-like catalytic activity. Conjugates with A₃, A₅, A₇, and 3A₃ anchors showed the highest levels of signals of bound labels on test strips, exceeding conjugates in sensitivity by up to nine times. These findings hold significant potential for broad application in bioanalytical systems. Full article

RNA is a promising nucleic acid-based biomolecule for various treatments because of its high efficacy, low toxicity, and the tremendous availability of targeting sequences. Nevertheless, RNA shows instability and has a short half-life in physiological environments such as the bloodstream in the presence of RNAase. Therefore, developing reliable delivery strategies is important for targeting disease sites and maximizing the therapeutic effect of RNA drugs, particularly in the field of immunotherapy. In this mini-review, we highlight two major approaches: (1) delivery vehicles and (2) chemical modifications. Recent advances in delivery vehicles employ nanotechnologies such as lipid-based nanoparticles, viral vectors, and inorganic nanocarriers to precisely target specific cell types to facilitate RNA cellular entry. On the other hand, chemical modification utilizes the alteration of RNA structures via the addition of covalent bonds such as N-acetylgalactosamine or antibodies (antibody–oligonucleotide conjugates) to target specific receptors of cells. The pros and cons of these technologies are enlisted in this review. We aim to review nucleic acid drugs, their delivery systems, targeting strategies, and related chemical modifications. Finally, we express our perspective on the potential combination of RNA-based click chemistry with adoptive cell therapy (e.g., B cells or T cells) to address the issues of short duration and short half-life associated with antibody–oligonucleotide conjugate drugs. Full article