-
The Journal of Organic Chemistry Jan 2021With a renewed and growing interest in therapeutic oligonucleotides across the pharmaceutical industry, pressure is increasing on drug developers to take more seriously...
With a renewed and growing interest in therapeutic oligonucleotides across the pharmaceutical industry, pressure is increasing on drug developers to take more seriously the sustainability ramifications of this modality. With 12 oligonucleotide drugs reaching the market to date and hundreds more in clinical trials and preclinical development, the current state of the art in oligonucleotide production poses a waste and cost burden to manufacturers. Legacy technologies make use of large volumes of hazardous reagents and solvents, as well as energy-intensive processes in synthesis, purification, and isolation. In 2016, the American Chemical Society (ACS) Green Chemistry Institute Pharmaceutical Roundtable (GCIPR) identified the development of greener processes for oligonucleotide Active Pharmaceutical Ingredients (APIs) as a critical unmet need. As a result, the Roundtable formed a focus team with the remit of identifying green chemistry and engineering improvements that would make oligonucleotide production more sustainable. In this Perspective, we summarize the present challenges in oligonucleotide synthesis, purification, and isolation; highlight potential solutions; and encourage synergies between academia; contract research, development and manufacturing organizations; and the pharmaceutical industry. A critical part of our assessment includes Process Mass Intensity (PMI) data from multiple companies to provide preliminary baseline metrics for current oligonucleotide manufacturing processes.
Topics: Drug Industry; Oligonucleotides; Solvents
PubMed: 33253568
DOI: 10.1021/acs.joc.0c02291 -
Beyond small molecules: targeting G-quadruplex structures with oligonucleotides and their analogues.Nucleic Acids Research Jul 2021G-Quadruplexes (G4s) are widely studied secondary DNA/RNA structures, naturally occurring when G-rich sequences are present. The strategic localization of G4s in genome... (Review)
Review
G-Quadruplexes (G4s) are widely studied secondary DNA/RNA structures, naturally occurring when G-rich sequences are present. The strategic localization of G4s in genome areas of crucial importance, such as proto-oncogenes and telomeres, entails fundamental implications in terms of gene expression regulation and other important biological processes. Although thousands of small molecules capable to induce G4 stabilization have been reported over the past 20 years, approaches based on the hybridization of a synthetic probe, allowing sequence-specific G4-recognition and targeting are still rather limited. In this review, after introducing important general notions about G4s, we aim to list, explain and critically analyse in more detail the principal approaches available to target G4s by using oligonucleotides and synthetic analogues such as Locked Nucleic Acids (LNAs) and Peptide Nucleic Acids (PNAs), reporting on the most relevant examples described in literature to date.
Topics: G-Quadruplexes; Genome, Human; Humans; Ligands; Nucleic Acid Heteroduplexes; Oligonucleotides; Peptide Nucleic Acids; Transcriptome
PubMed: 33978760
DOI: 10.1093/nar/gkab334 -
Biosensors Dec 2023Immunoassays based on antibodies as recognizing elements and enzymes as signal-generating modules are extensively used now in clinical lab diagnostics, food, and... (Review)
Review
Immunoassays based on antibodies as recognizing elements and enzymes as signal-generating modules are extensively used now in clinical lab diagnostics, food, and environmental analyses. However, the application of natural enzymes and antibodies has some drawbacks, such as relatively high manufacturing costs, thermal instability, and lot-to-lot variations that lower the reproducibility of results. Oligonucleotide aptamers are able to specifically bind their targets with high affinity and selectivity, so they represent a prospective alternative to protein antibodies for analyte recognition. Their main advantages include thermal stability and long shelf life, cost-efficient chemical synthesis, and negligible batch-to-batch variations. At the same time, a wide variety of non-protein peroxidase mimics are now available that show strong potential to replace protein enzymes. Here, we review and analyze non-protein biosensors that represent a nexus of these two concepts: aptamer-based sensors (aptasensors) with optical detection (colorimetric, luminescent, or fluorescent) based on different peroxidase mimics, such as DNAzymes, nanoparticles, or metal-organic frameworks.
Topics: Peroxidase; Prospective Studies; Reproducibility of Results; Peroxidases; Oligonucleotides; Biosensing Techniques; Antibodies; Aptamers, Nucleotide
PubMed: 38275302
DOI: 10.3390/bios14010001 -
Nature Chemistry Nov 2022Robotic systems for synthetic chemistry are becoming more common, but they are expensive, fixed to a narrow set of reactions, and must be used within a complex...
Robotic systems for synthetic chemistry are becoming more common, but they are expensive, fixed to a narrow set of reactions, and must be used within a complex laboratory environment. A portable system that could synthesize known molecules anywhere, on demand, and in a fully automated way, could revolutionize access to important molecules. Here we present a portable suitcase-sized chemical synthesis platform containing all the modules required for synthesis and purification. The system uses a chemical programming language coupled to a digital reactor generator to produce reactors and executable protocols based on text-based literature syntheses. Simultaneously, the platform generates a reaction pressure fingerprint, used to monitor processes within the reactors and remotely perform a protocol quality control. We demonstrate the system by synthesizing five small organic molecules, four oligopeptides and four oligonucleotides, in good yields and purities, with a total of 24,936 base steps executed over 329 h of platform runtime.
Topics: Oligonucleotides
PubMed: 36202987
DOI: 10.1038/s41557-022-01016-w -
RNA Biology Jan 2022From the early days of research on RNA biology and biochemistry, there was an interest to utilize this knowledge and RNA itself for therapeutic applications. Today, we... (Review)
Review
From the early days of research on RNA biology and biochemistry, there was an interest to utilize this knowledge and RNA itself for therapeutic applications. Today, we have a series of oligonucleotide therapeutics on the market and many more in clinical trials. These drugs - exploit different chemistries of oligonucleotides, such as modified DNAs and RNAs, peptide nucleic acids (PNAs) or phosphorodiamidate morpholino oligomers (PMOs), and different mechanisms of action, such as RNA interference (RNAi), targeted RNA degradation, splicing modulation, gene expression and modification. Despite major successes e.g. mRNA vaccines developed against SARS-CoV-2 to control COVID-19 pandemic, development of therapies for other diseases is still limited by inefficient delivery of oligonucleotides to specific tissues and organs and often prohibitive costs for the final drug. This is even more critical when targeting multifactorial disorders and patient-specific biological variations. In this review, we will present the evolution of complexity of oligonucleotide delivery methods with focus on increasing complexity of formulations from gymnotic delivery to bioconjugates and to lipid nanoparticles in respect to developments that will enable application of therapeutic oligonucleotides as drugs in personalized therapies.
Topics: Humans; Oligonucleotides; Biomimetics; Pandemics; SARS-CoV-2; Nanoparticles; RNA; COVID-19 Drug Treatment
PubMed: 36411594
DOI: 10.1080/15476286.2022.2147278 -
Cell Chemical Biology Mar 2020In a recent issue of Cell Chemical Biology, Gray et al. (2020) report an aptamer-based method to reversibly label and isolate EGF receptor-expressing cells from...
In a recent issue of Cell Chemical Biology, Gray et al. (2020) report an aptamer-based method to reversibly label and isolate EGF receptor-expressing cells from heterogeneous mixtures by cell sorting approaches. Subsequent treatment using complementary oligonucleotides restores full functionality of EGF receptors, highlighting the superiority of this method to antibody-based sorting.
Topics: Cell Movement; Ligands; Oligonucleotides; Protein Transport
PubMed: 32200913
DOI: 10.1016/j.chembiol.2020.02.006 -
Biochemistry Jan 2023Chemical biosensors are an increasingly ubiquitous part of our lives. Beyond enzyme-coupled assays, recent synthetic biology advances now allow us to hijack more complex... (Review)
Review
Chemical biosensors are an increasingly ubiquitous part of our lives. Beyond enzyme-coupled assays, recent synthetic biology advances now allow us to hijack more complex biosensing systems to respond to difficult to detect analytes, such as chemical small molecules. Here, we briefly overview recent advances in the biosensing of small molecules, including nucleic acid aptamers, allosteric transcription factors, and two-component systems. We then look more closely at a recently developed chemical sensing system, G protein-coupled receptor (GPCR)-based sensors. Finally, we consider the chemical sensing capabilities of the largest GPCR subfamily, olfactory receptors (ORs). We examine ORs' role in nature, their potential as a biomedical target, and their ability to detect compounds not amenable for detection using other biological scaffolds. We conclude by evaluating the current challenges, opportunities, and future applications of GPCR- and OR-based sensors.
Topics: Receptors, Odorant; Receptors, G-Protein-Coupled; Biosensing Techniques; Oligonucleotides; Nucleic Acids
PubMed: 36318941
DOI: 10.1021/acs.biochem.2c00486 -
Journal of Inorganic Biochemistry Oct 2023Two oligonucleotide conjugates sharing the same sequence but incorporating a different 5'-terminal organometallic moiety were synthesized, by either direct mercuration...
Two oligonucleotide conjugates sharing the same sequence but incorporating a different 5'-terminal organometallic moiety were synthesized, by either direct mercuration in solution or oximation with an organomercury aldehyde on solid support. The potential of these conjugates to serve as new type of artificial ribonucleases was tested with a complementary 2´-O-methyl-RNA target sequence featuring a single cleavable RNA phosphodiester linkage. Both organomercury oligonucleotides greatly outperformed their metal-free counterparts as well as the previously reported small molecule organomercury RNA cleaving agent in catalytic activity, providing an important proof-of-concept. Compared to state-of-the-art metal-dependent artificial ribonucleases, however, the observed activity was modest.
Topics: Aldehydes; Oligonucleotides; RNA; Ribonucleases
PubMed: 37480764
DOI: 10.1016/j.jinorgbio.2023.112331 -
Nucleic Acid Therapeutics Dec 2021Steric-blocking oligonucleotides (SBOs) are short, single-stranded nucleic acids designed to modulate gene expression by binding to RNA transcripts and blocking access...
Steric-blocking oligonucleotides (SBOs) are short, single-stranded nucleic acids designed to modulate gene expression by binding to RNA transcripts and blocking access from cellular machinery such as splicing factors. SBOs have the potential to bind to near-complementary sites in the transcriptome, causing off-target effects. In this study, we used RNA-seq to evaluate the off-target differential splicing events of 81 SBOs and differential expression events of 46 SBOs. Our results suggest that differential splicing events are predominantly hybridization driven, whereas differential expression events are more common and driven by other mechanisms (including spurious experimental variation). We further evaluated the performance of screens for off-target splicing events, and found an edit distance cutoff of three to result in a sensitivity of 14% and false discovery rate (FDR) of 99%. A machine learning model incorporating splicing predictions substantially improved the ability to prioritize low edit distance hits, increasing sensitivity from 4% to 26% at a fixed FDR of 90%. Despite these large improvements in performance, this approach does not detect the majority of events at an FDR <99%. Our results suggest that methods are currently of limited use for predicting the off-target effects of SBOs, and experimental screening by RNA-seq should be the preferred approach.
Topics: Alternative Splicing; Oligonucleotides; Oligonucleotides, Antisense; RNA; RNA Splicing; Transcriptome
PubMed: 34388351
DOI: 10.1089/nat.2020.0921 -
Angewandte Chemie (International Ed. in... Jul 2021We developed a new approach to selectively modify native proteins in their biological environment using electrophilic covalent aptamers. These aptamers are generated...
We developed a new approach to selectively modify native proteins in their biological environment using electrophilic covalent aptamers. These aptamers are generated through introduction of a proximity-driven electrophile at specific nucleotide sites. Using thrombin as a proof-of-concept, we demonstrate that covalent aptamers can selectively transfer a variety of functional handles and/or irreversibly crosslink to the target protein. This approach offers broad programmability and high target specificity. Furthermore, it addresses issues common to aptamers such as instability towards endogenous nucleases and residence times during target engagement. Covalent aptamers are new tools that enable specific protein modification and sensitive protein detection. Moreover, they provide prolonged, nuclease-resistant enzyme inhibition.
Topics: Aptamers, Nucleotide; Biosensing Techniques; Proteins; SELEX Aptamer Technique; Staining and Labeling; Thrombin
PubMed: 33928724
DOI: 10.1002/anie.202101174