-
Expert Review of Proteomics 2024Development of new methods is essential to make great leaps in science, opening up new avenues for research, but the process behind method development is seldom... (Review)
Review
INTRODUCTION
Development of new methods is essential to make great leaps in science, opening up new avenues for research, but the process behind method development is seldom described.
AREAS COVERED
Over the last twenty years we have been developing several new methods, such as in situ PLA, proxHCR, and MolBoolean, using oligonucleotide-conjugated antibodies to visualize protein-protein interactions. Herein, we describe the rationale behind the oligonucleotide systems of these methods. The main objective of this paper is to provide researchers with a description on how we thought when we designed those methods. We also describe in detail how the methods work and how one should interpret results.
EXPERT OPINION
Understanding how the methods work is important in selecting an appropriate method for your experiments. We also hope that this paper may be an inspiration for young researchers to enter the field of method development. Seeing a problem is a motivation to develop a solution.
Topics: Humans; Oligonucleotides; Antibodies
PubMed: 38363709
DOI: 10.1080/14789450.2024.2318565 -
Molecules (Basel, Switzerland) Jul 2023Phosphorodiamidate morpholinos (PMOs) are known as premier gene knockdown tools in developmental biology. PMOs are usually 25 nucleo-base-long morpholino subunits with a... (Review)
Review
Phosphorodiamidate morpholinos (PMOs) are known as premier gene knockdown tools in developmental biology. PMOs are usually 25 nucleo-base-long morpholino subunits with a neutral phosphorodiamidate linkage. PMOs work via a steric blocking mechanism and are stable towards nucleases' inside cells. PMOs are usually synthesized using phosphoramidate P(V) chemistry. In this review, we will discuss the synthesis of PMOs, phosphoroamidate morpholinos (MO), and thiophosphoramidate morpholinos (TMO).
Topics: Morpholinos; Organophosphorus Compounds; Oligonucleotides, Antisense
PubMed: 37513252
DOI: 10.3390/molecules28145380 -
Bioorganic Chemistry Dec 2023Nucleic acid represents the ideal drug candidate for protein targets that are hard to target or against which drug development is not easy. Peptide nucleic acids (PNAs)... (Review)
Review
Nucleic acid represents the ideal drug candidate for protein targets that are hard to target or against which drug development is not easy. Peptide nucleic acids (PNAs) are synthesized by attaching modified peptide backbones generally derived from repetitive N-2-aminoethyl glycine units in place of the regular phosphodiester backbone and represent synthetic impersonator of nucleic acids that offers an exciting research field due to their fascinating spectrum of biotechnological, diagnostic and potential therapeutic applications. The semi-rigid peptide nucleic acid backbone serves as a nearly-perfect template for attaching complimentary base pairs on DNA or RNA in a sequence-dependent manner as described by Watson-Crick models. PNAs and their analogues are endowed with exceptionally high affinity and specificity for receptor sites, essentially due to their polyamide backbone's uncharged and flexible nature. The present review compiled various strategies to modify the polypeptide backbone for improving the target selectivity and stability of the PNAs in the body. The investigated biological activities carried out on PNAs have also been summarized in the present review.
Topics: Peptide Nucleic Acids; RNA; DNA; Peptides; Binding Sites
PubMed: 37748328
DOI: 10.1016/j.bioorg.2023.106860 -
Nature Communications Dec 2023Off-target interactions between antisense oligonucleotides (ASOs) with state-of-the-art modifications and biological components still pose clinical safety liabilities....
Off-target interactions between antisense oligonucleotides (ASOs) with state-of-the-art modifications and biological components still pose clinical safety liabilities. To mitigate a broad spectrum of off-target interactions and enhance the safety profile of ASO drugs, we here devise a nanoarchitecture named BRace On a THERapeutic aSo (BROTHERS or BRO), which is composed of a standard gapmer ASO paired with a partially complementary peptide nucleic acid (PNA) strand. We show that these non-canonical ASO/PNA hybrids have reduced non-specific protein-binding capacity. The optimization of the structural and thermodynamic characteristics of this duplex system enables the operation of an in vivo toehold-mediated strand displacement (TMSD) reaction, effectively reducing hybridization with RNA off-targets. The optimized BROs dramatically mitigate hepatotoxicity while maintaining the on-target knockdown activity of their parent ASOs in vivo. This technique not only introduces a BRO class of drugs that could have a transformative impact on the extrahepatic delivery of ASOs, but can also help uncover the toxicity mechanism of ASOs.
Topics: Male; Humans; Oligonucleotides, Antisense; RNA; Protein Binding; Peptide Nucleic Acids; Nucleic Acid Hybridization; Phosphorothioate Oligonucleotides
PubMed: 38042877
DOI: 10.1038/s41467-023-43714-0 -
Organic Letters Feb 2024A method for phosphorylating oligonucleotides using a thermosensitive "trigger" is hereby presented. The recovery of the phosphate specifically takes place under neutral...
A method for phosphorylating oligonucleotides using a thermosensitive "trigger" is hereby presented. The recovery of the phosphate specifically takes place under neutral conditions when subjected to an elevated temperature. Two identical thermolabile protecting groups are differentially removed with the initial release occurring swiftly and the second at a more gradual pace. The delayed deprotection of the second group led to the development of a method for the purification of 5'-phosphorylated oligonucleotides. Microwave irradiation enables the rapid attainment of complete deprotection, in contrast to conventional heating methods.
Topics: Oligonucleotides; Microwaves; Phosphorylation; Phosphates
PubMed: 38307039
DOI: 10.1021/acs.orglett.3c03924 -
Bioorganic Chemistry Nov 2023Solid-phase synthesis is, to date, the preferred method for the manufacture of oligonucleotides, in quantities ranging from a few micrograms for research purposes to... (Review)
Review
Solid-phase synthesis is, to date, the preferred method for the manufacture of oligonucleotides, in quantities ranging from a few micrograms for research purposes to several kilograms for therapeutic or commercial use. But for large-scale oligonucleotide manufacture, scaling up and hazardous waste production pose challenges that necessitate the investigation of alternate synthetic techniques. Despite the disadvantages of glass supports, using soluble supports as a substitute presents difficulties because of their high overall yield and complex purification steps. To address these challenges, various independent approaches have been developed; however, other problems such as insufficient cycle efficiency and synthesis of oligonucleotide chains of desired length continue to exist. In this study, we present a review of the current developments, advantages, and difficulties of recently reported alternatives to supports based on controlled pore glass, and discuss the importance of a support choice to resolve issues arising during oligonucleotide synthesis.
Topics: Oligonucleotides; Nucleic Acids
PubMed: 37660625
DOI: 10.1016/j.bioorg.2023.106806 -
ACS Macro Letters Sep 2023We present a facile and adaptable method to purify and isolate DNA-polymer conjugates from different uncharged homo, random, or block copolymer families. Anion exchange...
We present a facile and adaptable method to purify and isolate DNA-polymer conjugates from different uncharged homo, random, or block copolymer families. Anion exchange chromatography is used to separate the reaction solution and retrieve the excess unreacted polymer and oligonucleotide. The stationary phase has a high efficiency (25 nmol of DNA per run), facilitating the purification of large batches without compromising the peak shape and resolution. To demonstrate the versatility of this method, different types of polymers, including acrylates, methacrylates, and acrylamides containing hydrophilic and hydrophobic blocks, were purified with high yields. Additionally, DNA-polymer conjugates with various DNA block lengths were also successfully purified, further highlighting the broad applicability of this method.
Topics: Humans; DNA; Oligonucleotides; Acrylamides; Acrylates; Polymers
PubMed: 37656875
DOI: 10.1021/acsmacrolett.3c00371 -
Analytical Chemistry Oct 2023The -cleavage activity of CRISPR/Cas12a has been widely used in biosensing. However, many CRISPR/Cas12a-based biosensors, especially those that work in "on-off-on" mode,...
The -cleavage activity of CRISPR/Cas12a has been widely used in biosensing. However, many CRISPR/Cas12a-based biosensors, especially those that work in "on-off-on" mode, usually suffer from high background and thus impossible intracellular application. Herein, this problem is efficiently overcome by elaborately designing the activator strand (AS) of CRISPR/Cas12a using the "RESET" effect found by our group. The activation ability of the as-designed AS to CRISPR/Cas12a can be easily inhibited, thus assuring a low background for subsequent biosensing applications, which not only benefits the detection sensitivity improvement of CRISPR/Cas12a-based biosensors but also promotes their applications in live cells as well as makes it possible to design high-performance biosensors with greatly improved flexibility, thus achieving the analysis of a wide range of targets. As examples, by using different strategies such as strand displacement, strand cleavage, and aptamer-substrate interaction to reactivate the inhibited enzyme activity, several CRISPR/Cas12a-based biosensing systems are developed for the sensitive and specific detection of different targets, including nucleic acid (miR-21), biological small molecules (ATP), and enzymes (hOGG1), giving the detection limits of 0.96 pM, 8.6 μM, and 8.3 × 10 U/mL, respectively. Thanks to the low background, these biosensors are demonstrated to work well for the accurate imaging analysis of different biomolecules in live cells. Moreover, we also demonstrate that these sensing systems can be easily combined with lateral flow assay (LFA), thus holding great potential in point-of-care testing, especially in poorly equipped or nonlaboratory environments.
Topics: CRISPR-Cas Systems; Biological Assay; Image Processing, Computer-Assisted; Nucleic Acids; Oligonucleotides; Biosensing Techniques
PubMed: 37819747
DOI: 10.1021/acs.analchem.3c03131 -
Bioconjugate Chemistry Aug 2023Nucleic acid-based medicines and vaccines are becoming an important part of our therapeutic toolbox. One key genetic medicine is antisense oligonucleotides (ASOs), which...
Nucleic acid-based medicines and vaccines are becoming an important part of our therapeutic toolbox. One key genetic medicine is antisense oligonucleotides (ASOs), which are short single-stranded nucleic acids that downregulate protein production by binding to mRNA. However, ASOs cannot enter the cell without a delivery vehicle. Diblock polymers containing cationic and hydrophobic blocks self-assemble into micelles that have shown improved delivery compared to linear nonmicelle variants. Yet synthetic and characterization bottlenecks have hindered rapid screening and optimization. In this study, we aim to develop a method to increase throughput and discovery of new micelle systems by mixing diblock polymers together to rapidly form new micelle formulations. We synthesized diblocks containing an -butyl acrylate block chain extended with cationic moieties amino ethyl acrylamide (A), dimethyl amino ethyl acrylamide (D), or morpholino ethyl acrylamide (M). These diblocks were then self-assembled into homomicelles (A100, D100, and M100)), mixed micelles comprising 2 homomicelles (MixR%+R'%), and blended diblock micelles comprising 2 diblocks blended into one micelle (BldR%R'%) and tested for ASO delivery. Interestingly, we observed that mixing or blending M with A (BldA50M50 and MixA50+M50) did not improve transfection efficiency compared to A100; however, when M was mixed with D, there was a significant increase in transfection efficacy for the mixed micelle MixD50+M50 compared to D100. We further examined mixed and blended D systems at different ratios. We observed a large increase in transfection and minimal change in toxicity when M was mixed with D at a low percentage of D incorporation in mixed diblock micelles (i.e., BldD20M80) compared to D100 and MixD20+M80. To understand the cellular mechanisms that may result in these differences, we added proton pump inhibitor Bafilomycin-A1 (Baf-A1) to the transfection experiments. Formulations that contain D decreased in performance in the presence of Baf-A1, indicating that micelles with D rely on the proton sponge effect for endosomal escape more than micelles with A. This result supports our conclusion that M is able to modulate transfection of D, but not with A. This research shows that polymer blending in a manner similar to that of lipids can significantly boost transfection efficiency and is a facile way to increase throughput of testing, optimization, and successful formulation identification for polymeric nucleic acid delivery systems.
Topics: Micelles; Oligonucleotides, Antisense; Polymers; Oligonucleotides; Acrylamides
PubMed: 37437196
DOI: 10.1021/acs.bioconjchem.3c00186 -
Chemical Communications (Cambridge,... Jul 2023The most significant challenge for nucleic acid drug development is their delivery across the cell membrane. Herein, we harness the reversible binding between boronic...
The most significant challenge for nucleic acid drug development is their delivery across the cell membrane. Herein, we harness the reversible binding between boronic acids and cell surface glycans to aid in the cellular delivery of synthetic oligonucleotides. We install the artificial nucleotide 5-dihydroxyboryluridine (5boU) in a site-specific manner within druglike antisense oligonucleotides and demonstrate that these boronate-containing nucleic acids have enhanced cytosolic penetration and splice-correcting activity compared to non-boronate analogs. Strategic incorporation of 5boU is a simple, modular, and potentially general means of enhancing cellular delivery of therapeutic nucleic acids.
Topics: Oligonucleotides, Antisense; Oligonucleotides; Nucleic Acids
PubMed: 37345964
DOI: 10.1039/d3cc01945d