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Oligonucleotides are essential for gene regulation, expression, and disease biomarker identification, yet their small size presents challenges due to lower abundance and increased susceptibility to degradation in biological samples. Addressing these challenges, a novel approach was developed for effective oligonucleotide extraction, consisting of a commercially available nitrocellulose (NC) membrane non-covalently modified with a combination of single-walled carbon nanotubes (SWCNTs) and polyethylene glycol (PEG) aminated reduced graphene oxide (GA). The membrane was evaluated for the extraction of a fluorescent labelled single-stranded deoxyribonucleic acid (ssDNA), with fewer than 30 nucleotides, from complex solutions containing various ionic species (MnCl 2 , MgCl 2 , and MnCl 2 /MgCl 2 ). Fourier transform infrared spectroscopy confirmed successful modification, revealing characteristic peaks of NC, SWCNT, and GA. Raman spectroscopy and X-ray photoelectron spectroscopy showed distinctive changes after the membrane interaction with divalent cations and ssDNA. Scanning electron microscopy revealed morphological changes in the SWCNTs/GA-NC hybrid membrane, showing a smoother surface compared to the porous structure of the unmodified NC membrane. Wettability assays indicated hydrophobic properties for the SWCNT/GA-NC hybrid membrane, with a water contact angle exceeding 110°, contrasting with the hydrophilic nature of the NC membrane, which exhibits a contact angle of 26.7°. Optimal performance of the SWCNTs/GA-NC hybrid membrane was observed when incubated in MgCl 2 , demonstrating the highest fluorescence emission at approximately 670 relative fluorescence units. This corresponded to the extraction of approximately 781 pg (≈ 16%) of the total oligo-DNA, highlighting the enhanced efficacy of the hybrid material compared to the unmodified NC membrane, which extracted only 318 pg (≈ 7%) of oligo-DNA., Competing Interests: Declarations. Use of generative AI and AI-assisted tools: The authors used ChatGPT by OpenAI in order to improve the readability and language of the manuscript. The authors reviewed and edited the generated output and take full responsibility for the content of the published article. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Splice-switching oligonucleotides (SSOs) can restore protein functionality in pathologies and are promising tools for manipulating the RNA-splicing machinery. Delivery vectors can considerably improve SSO functionality in vivo and allow dose reduction, thereby addressing the challenges of RNA-targeted therapeutics. Here, we report a biocompatible SSO nanocarrier, based on redox-responsive disulfide cross-linked low-molecular-weight linear polyethylenimine (cLPEI), for overcoming multiple biological barriers from subcellular compartments to en-route serum stability and finally in vivo delivery challenges. Intracellularly responsive cross-links of cLPEI significantly accelerated the endosomal escape and offered efficient SSO release to the cell's nucleus, thereby leading to high splice correction in vitro. In vivo performance of cLPEI-SSOs was investigated in a novel transgenic mouse model for splice correction, spatiotemporal tracking of SSO delivery in wild-type mice, and biodistribution in a colorectal cancer peritoneal metastasis model. A single intravenous application of 5 mg kg -1 cLPEI-SSOs induced splice correction in liver, lung, kidney, and bladder, giving functional protein, which was validated by RT-PCR. Near-infrared (NIR) fluorescence imaging and X-ray computed tomography revealed improved organ retention and reduced renal excretion of SSOs. NIR microscopy demonstrated the accumulation of SSOs in angiogenic tumors within the pancreas. Successful nuclear delivery of SSOs was observed in the hepatocytes. Thus, cLPEI nanocarriers resulted in highly efficient splice correction in vivo, highlighting the critical role of the enhanced SSO bioavailability.

Nucleic acids, used as therapeutics to silence disease-related genes, offer significant advantages over small molecule drugs: they provide high specificity, the ability to target "undruggable" molecules, and adaptability to a wide range of disease phenotypes. However, their instability in biological media, as well their rapid clearance from the organism limit their applicability, necessitating the use of nanocarriers to overcome these challenges. Among these strategies, spherical nucleic acids (SNA)-composed of a densely packed corona of oligonucleotides around a nanoparticle-have emerged as a powerful tool, in particular when self-assembled from DNA amphiphiles. This non-covalent strategy however has caveats, especially when it comes to stability in complex biological media, where these SNAs disassemble in contact to serum proteins. Here, we developed highly fluorinated DNA amphiphiles that readily self-assemble into SNAs and have tunable stability profiles in biological media. They are made of branched fluorinated moieties with potentially improved biodegradability as compared to their linear counterparts. Depending on the number of fluorophilic interactions, the self-assembled SNAs can have excellent serum stabilities-up to days-and readily deliver nucleic acid therapeutics for gene silencing applications. These systems show great potential as promising candidates for nucleic acid-based therapies., (© 2024 The Author(s). Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Background: Infections from the hepatitis B virus (HBV) are a major risk factor for hepatocellular carcinoma, one of the most common types of liver cancer. Circulating cell-free DNA (ccfDNA) in human plasma can be used as a non-invasive biomarker for diagnosing HBV-related liver diseases. The isolation of target ccfDNA sequences is often challenging due to the co-extraction of highly abundant non-target DNA from samples. Ion-tagged oligonucleotide (ITO) probes coupled with magnetic ionic liquids (MIL) support have emerged as a promising methodology for sequence-specific DNA extractions to address challenges associated with solid supports such as streptavidin-coated magnetic bead., Results: ITOs, disubstituted ion-tagged oligonucleotide (DTO), and ion-tagged disubstituted oligonucleotide (ITDO) probes featuring various substituents including linear alkyl, branched alkyl, and perfluoroalkyl moieties were examined to enhance hydrophobic and fluorophilic interactions between the probes and a trihexyl(tetradecyl)phosphonium manganese(II) hexafluoroacetylacetonate ([P 66614 + ][Mn(hfacac) 3 - ]) MIL support. The ITO-MIL approach was optimized by adjusting the annealing temperature (45 °C), molar ratio of target DNA to ITO probe (1:100), and ionic strength (200 mM NaCl) to maximize the extraction of target DNA. The 3-allyl-1-(23-bis(pentylthio)propyl)imidazolium bromide ([AI(C 5 S) 2 + ][Br - ])-ITO probe featuring a branched alkyl substituent yielded a higher loading efficiency (48.05 ± 6.72 %) due to increased hydrophobic ITO-MIL interactions, compared to that of 24.57 ± 4.40 % for the 3-allyl-1-(3-(pentylthio)propyl)imidazolium bromide ([AIC 5 S + ][Br - ])-ITO probe with a linear alkyl substituent. The high affinity of ITO-MILinteractions minimized the loss of probe during successive wash steps, yielding an approximate 10-fold greater amount of extracted target DNA using the [AI(C 5 S) 2 + ][Br - ])-ITO probe compared to [AIC 5 S + ][Br - ])-ITO probe., Significance: This study provides novel synthetic approaches for tailoring probe substituents using thiol-ene and thiol-yne "click" chemistry. Hydrophobic and fluorophilic ITO-MIL interactions were systematically investigated. Optimal conditions for the ITO-MIL method significantly improved the amount of extracted DNA. The ITO-MIL method using the [AI(C 5 S) 2 + ][Br - ]-HBV-ITO probe demonstrated selective extraction of target HBV DNA in both DI water and diluted human plasma containing a high abundance of background DNA., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest in this work., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Despite the unique advantage of the isothermal exponential amplification reaction (EXPAR) for the rapid detection of short nucleic acids, it severely suffers from the drawback of sequence-dependent amplification bias, mainly arising from the secondary structures of the EXPAR template under the commonly used reaction temperature (55 °C). As such, the limits of detection (LOD) for different target sequences may vary considerably from aM to nM. Here we report a sequence-generic exponential amplification reaction (SG-EXPAR) that eliminates sequence-dependent amplification bias and achieves similar amplification performance for different targets with generally sub-fM LODs. The assay innovatively employs a thermophilic nicking enzyme that allows SG-EXPAR to work efficiently at higher temperatures (60-70 °C) while eliminating the secondary structures of the templates, which is the basis for eliminating the amplification bias. Furthermore, we increased the probability of trigger/template binding through rational modification of the locked nucleic acids and template optimization, further ensuring the high amplification efficiency for various targets. According to these critical principles, we have developed an automated design platform that allows nonspecialists to obtain the optimal SG-EXPAR template for any desired sequence. The robust performance of the proposed methodology was demonstrated by quantifying microRNA, SARS-CoV-2, monkeypox virus, and HPV B19 at the 1 fM level without sequence screening. SG-EXPAR significantly expands the potential applications of EXPAR and facilitates the development of reliable point-of-care nucleic acid assays.

Understanding the interactions between lipid membranes and nucleotide drugs is crucial for nucleic acid therapy. Although several methods have been employed to evaluate nucleotide-lipid membrane interactions, these interactions can be complex; this complexity arises from how external factors, such as ionic strength or temperature, influence the lipid membrane's overall properties. In this study, we prepared a lipid membrane-immobilized monolithic silica (LMiMS) column for high-performance liquid chromatography (HPLC) analysis to understand interactions between the lipid membrane and nucleic acid. First, the Raman shift, zeta potential, fluidity, and polarity of the LMiMS-column membrane were analyzed and compared to dispersed liposomes (not immobilized) with the same lipid composition. The results indicated that the column can effectively imitate the temperature-dependent properties of the lipid membrane, suggesting that the immobilized lipid membrane can be used as a model of dispersed liposomal membranes. Subsequently, HPLC was performed to analyze the interaction between the lipid membrane and oligonucleotides. The retention factor k was determined as an interaction factor between the LMiMS column and nucleic acid models (poly 10, 25, and 50mer dC) at various temperatures and mobile phase salt concentrations. The results revealed that changes in membrane interaction were significant by the phase state and salt concentration. Further analysis of the retention factor k showed that the interaction is weak below the phase transition temperature but strong above the phase transition temperature. The results indicate that membrane properties (rigidity and polarity) are also related to membrane interaction, which can be evaluated with the LMiMS column. This analytical method can provide a new perspective on estimating the interactions between target molecules and the lipid membrane through their temperature-induced dynamics. From these results, our method could be useful for analyzing lipid membrane-oligonucleotide interactions.

Gapmer-type antisense oligonucleotides (ASOs) are an emerging class of therapeutic agents that directly inhibit pathogenic mRNA. In this study, three new 4'- C -substituted thymidine analogs were generated using a synthetic strategy recently established by our group, namely, 4'- C -( N -ethyl) aminoethyl (4'-EAE-T), 4'- C -( N -butyl) aminoethyl (4'-BAE-T), and 4'- C -( N -octyl) aminoethyl (4'-OAE-T). Their properties were evaluated and compared with those of previously reported analogs, including 4'- C -aminoethyl (4'-AE-T) and 4'- C -( N -methyl) aminoethyl (4'-MAE-T). The novel nucleoside analogs were subsequently incorporated into gapmer-type ASOs featuring phosphorothioate (PS) linkages and locked nucleic acids (LNAs) in the wing regions. The incorporation of 4'-EAE-T and 4'-BAE-T analogs resulted in RNA binding affinities similar to that of the previously reported 4'-MAE-T analog, whereas a marked decrease in RNA affinity was noted for 4'-OAE-T, however, this reduction was mitigated when combined with other chemical modifications. Furthermore, the structural modifications conferred enhanced nuclease resistance under bovine serum conditions, with 4'-EAE-T resulting in the highest stability, followed by 4'-BAE-T and 4'-OAE-T. Additionally, oligonucleotides modified with the developed analogs preserved their RNase H cleavage susceptibility, albeit inducing minor alterations in the cleavage pattern. Finally, the oligonucleotides were applied in a gene silencing experiment targeting the KRAS gene, conducted without the use of transfection agents, displaying gene silencing activities comparable to that of the control, with the exception of the 4'-OAE-modified nucleotide, which exhibited low activity.

Chemically modified oligonucleotides have garnered significant attention in medicinal chemistry, chemical biology, and synthetic biology due to their enhanced stability in vivo compared to naturally occurring oligonucleotides. However, current methods for synthesizing modified nucleosides, particularly at the C2'-position, are limited in terms of efficiency, modularity, and selectivity. Herein, we report a new approach for the synthesis of highly functionalized C2'-α-aryl/alkenyl nucleosides via an electrochemical nickel-catalyzed cross-coupling of 2'-bromo nucleosides with a variety of (hetero)aryl and alkenyl iodides. This method affords a diverse array of C2'- α-aryl/alkenyl nucleosides with excellent stereoselectivity, broad substrate scope, and good functional group compatibility. We further synthesized oligonucleotides incorporating C2'-aryl-modified thymidine moieties and demonstrated that their annealed double-stranded DNAs exhibit decreased melting temperatures (Tm). Additionally, oligonucleotides with C2'-aryl modifications at the 3' end showed enhanced resistance to 3'-exonuclease degradation and C2'-aryl modifications did not impede the cellular uptake process, highlighting the potential of these modified oligonucleotides for therapeutic applications., (© 2024 Wiley-VCH GmbH.)

Time-resolved small-angle X-ray experiments are reported here that capture and quantify a previously unknown rapid collapse of the unfolded oligonucleotide as an early step in the folding of hybrid 1 and hybrid 2 telomeric G-quadruplex structures. The rapid collapse, initiated by a pH jump, is characterized by an exponential decrease in the radius of gyration from 24.3 to 12.6 Å. The collapse is monophasic and is complete in <600 ms. Additional hand-mixing pH-jump kinetic studies show that slower kinetic steps follow the collapse. The folded and unfolded states at equilibrium were further characterized by SAXS studies and other biophysical tools, showing that G4 unfolding was complete at alkaline pH, but not in LiCl solution as is often claimed. The SAXS Ensemble Optimization Method analysis reveals models of the unfolded state as a dynamic ensemble of flexible oligonucleotide chains with a variety of transient hairpin structures. These results suggest a G4 folding pathway in which a rapid collapse, analogous to molten globule formation seen in proteins, is followed by a confined conformational search within the collapsed particle to form the native contacts ultimately found in the stable folded form., (© The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Synthetic ssDNA oligonucleotides hold great potential for various applications, including DNA aptamers, DNA digital data storage, DNA origami, and synthetic genomes. In these contexts, precise control over the synthesis of the ssDNA strands is essential for generating combinatorial sequences with user-defined parameters. Desired features for creating synthetic DNA oligonucleotides include easy manipulation of DNA strands, effective detection of unique DNA sequences, and a straightforward mechanism for strand elongation and termination. In this study, we present a split-and-pool method for generating synthetic DNA oligonucleotides on nanoparticles, enabling the creation of scalable combinatorial libraries. Our approach involves coupling DNA to nanoparticles, ligating double-digested fragments for orientation-specific synthesis, and attaching a final single-digested fragment to ensure strand termination. We assess the quality of our method by characterizing both the DNA and the nanoparticles used as solid supports, confirming that our method produces scalable, combinatorial nanoparticle-bound ssDNA libraries with controllable strand lengths.

Oligonucleotides are a rapidly emerging class of therapeutics. Their most well-known examples are informational drugs that modify gene expression by binding mRNA. Despite inducing proximity between biological machinery and mRNA when applied to modulating gene expression, oligonucleotides are not typically labeled as "proximity-inducing" in literature. Yet, they have recently been explored as building blocks for multispecific proximity-inducing drugs (MPIDs). MPIDs are unique because they can direct endogenous biological machinery to destroy targeted molecules and cells, in contrast to traditional drugs that inhibit only their functions. The unique mechanism of action of MPIDs has enabled the targeting of previously "undruggable" molecular entities that cannot be effectively inhibited. However, the development of MPIDs must ensure that these molecules will selectively direct a potent, destruction-based mechanism of action toward intended targets over healthy tissues to avoid causing life-threatening toxicities. Oligonucleotides have emerged as promising building blocks for the design of MPIDs because they are sequence-controlled molecules that can be rationally designed to program multispecific binding interactions. In this Review, we examine the emergence of oligonucleotide-containing MPIDs in the proximity induction space, which has been dominated by antibody and small molecule MPID modalities. Moreover, examples of oligonucleotides developed as MPID candidates in immunotherapy and protein degradation are discussed to demonstrate the utility of oligonucleotides in expanding the scope and selectivity of the MPID toolbox. Finally, we discuss the utility of programming "AND" gates into oligonucleotide scaffolds to encode conditional responses that have the potential to be incorporated into MPIDs, which can further enhance their selectivity, thus increasing the scope of this drug category.

Development of robust oligonucleotide-based probe technologies, capable of recognizing specific regions of double-stranded DNA (dsDNA) targets, continues to attract considerable attention due to the promise of tools for modulation of gene expression, diagnostic agents, and new modalities against genetic diseases. Our laboratory pursues the development of various strand-invading probes. These include Invader probes, i.e. , double-stranded oligonucleotide probes with one or more +1 interstrand zipper arrangements of intercalator-functionalized nucleotides like 2'- O -(pyren-1-yl)methyl-RNA monomers, and chimeric Invader/γPNA probes, i.e. , heteroduplex probes between individual Invader strands and complementary γPNA strands. Here we report on the biophysical properties and dsDNA-recognition characteristics of a new class of chimeric probes-chimeric Invader/LNA probes-which are comprised of densely modified Invader strands and fully modified complementary LNA strands. The chimeric Invader/LNA probes form labile and distorted heteroduplexes, due to an apparent incompatibility between intercalating pyrene moieties and LNA strands. In contrast, the individual Invader and LNA strands form very stable duplexes with complementary DNA, which provides the driving force for near-stoichiometric recognition of model double-stranded DNA targets with single base-pair accuracy. The distinctive properties of chimeric Invader/LNA probes unlock exciting possibilities in molecular biology, and diagnostic and therapeutic fields.

Oligonucleotides are currently one of the most rapidly advancing classes of therapeutic modalities. Understanding critical quality attributes, such as the impurity profile, stability, potential metabolites, and sequence conformity, is the key to their ultimate success. To obtain the information presented above, liquid chromatography-mass spectrometry (LC-MS) is often employed. However, data interpretation can be challenging due to multiple charge states, unknown species, chemical noises, and overlapping isotope envelopes (OIEs) of distinct but unresolved species. To address these challenges, we have developed an MS-platform agnostic, multifunctional R package and a web-tool for automated and interactive MS and MS 2 data analysis, OligoDistiller . From the MS spectrum of a complex mixture of two synthetic strands, our tool OligoDistiller annotated and quantified 45 oligonucleotide-related features including 13 unknown impurities and 6 OIEs, which all together explained 90.8% of the detected peaks. Also, major product ions were assigned from the MS 2 spectrum of a 47-mer DNA strand, covering 80.3% of the oligonucleotide sequence. We provide not only diverse isotope quality metrics for each annotated feature but also an interactive data review module allowing for direct inspection of the part of raw spectrum linked to a selected feature. This tool OligoDistiller is freely available at https://github.com/daniellyz/OligoDistiller.

The need for accurate and simultaneous diagnosis of multiple respiratory infectious diseases has become increasingly critical due to ongoing viral mutations and the similarity of symptoms among various viruses. Here, we have advanced our detection capabilities by developing a multiplex lateral flow immunoassay (LFA) platform that integrates oligonucleotides and antibodies, enabling the simultaneous detection of five respiratory viruses: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Influenza A (FluA), Influenza B (FluB), Respiratory syncytial virus (RSV), and Adenovirus (ADV), on a single membrane. By applying the oligonucleotide and antibody-conjugated AuNPs, the platform enables highly sensitive and specific detection. In addition, signal amplification using RPA70A-conjugated gold nanoparticles that developed in the previous study can further be applied optionally for low-concentration biomarkers. Our interferences and cross-reactivity tests confirmed that these complexes do not produce false positives, substantiating the assay's utility in clinical settings. This platform, therefore, provides a robust solution for the precise and rapid diagnosis of complex viral infections, positioning it as suitable for application in pandemic response scenarios.

Three critical advances in simplifying the adoption of P(V)-based stereopure, phosphorothioate-containing oligonucleotide synthesis are reported. A more inexpensive phosphorus-sulfur incorporation reagent ( Ψ Br ) is introduced, a robust linker system was developed, and a systematic study of common nucleobase protecting groups was performed to significantly reduce the barrier to adoption of this technology.

Although glycosidic bonds in purines typically involve the N9 position, the chemical synthesis of adenosine produces N7-ribofuranosyladenine (7A) as a kinetically favorable ribosylation product. Similarly, in the synthesis of LNA-adenosine (AL), a minor product, N7-LNA-adenosine (7AL), is observed. While extensive research has focused on investigating the properties of N9-regioisomers of adenosine, 7A has been largely overlooked and considered as a side-product. In this study, we conducted comprehensive experimental and computational investigations to elucidate the structural and thermodynamic properties of 7A and 7AL. Our results reveal that 7A and 7AL primarily enhance the thermodynamic stability of 1 × 1 mismatches when paired with purines but decrease stability when paired with pyrimidines. Utilizing nuclear magnetic resonance and computational techniques, we discovered that 1 × 1 7A:A and 7AL:A prefer anti-anti conformations, while 1 × 1 7A:G and 7AL:G prefer syn-anti orientations, both forming two hydrogen bond states, resulting in enhanced duplex stabilities. Altogether, these findings underscore the unique properties of 7A and 7AL when incorporated in RNA, which could advance structure-based RNA studies and potentially be utilized to modulate binding affinity, selectivity and biostability of RNA molecules., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Ion-pair reversed-phase liquid chromatography (IP-RPLC) is clearly recognized as the gold standard for analyzing therapeutic oligonucleotides (ONs). Recent studies have shown that ONs exhibit an on-off retention behavior in IP-RPLC, meaning that minor changes in acetonitrile (ACN) proportion can significantly impact retention. However, this behavior was initially demonstrated with only a single mobile phase condition. The aim of this study is to gain a deeper understanding of ON elution behavior by measuring the S values (slope of the retention model, log k vs.%ACN) across a broad range of mobile phase conditions. We systematically calculated the S values for both a 20-mer and 100-mer model ON under various conditions, including different IP reagents, IP concentrations, mobile phase pH, column temperatures, and two different buffering acids. We demonstrated that these mobile phase conditions impact the S values in the following order: IP hydrophobicity > IP concentration > column temperature > buffering acid > mobile phase pH. The main explanation for this trend is that mobile phase conditions that reduce the ion-pair retention mechanism (such as low IP hydrophobicity or concentration) will enhance the impact of% ACN on retention, leading to higher S values. In the second part of the study, this knowledge was used to develop ultra-fast separations for two therapeutic oligonucleotides: a 20-mer antisense oligonucleotide (ASO) without phosphorothioate (PS) modifications and a large single guide RNA (sgRNA) that includes certain PS modifications. The mobile phase conditions were optimized to maximize S values, while preventing the separation of diastereomers. It is important to notice that an S-value of at least 30 is required to benefit from the use of ultra-short columns. This approach allows the successful separation of the main species (ASO and sgRNA) and related impurities in less than one minute using a 5 mm length column., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: This work was partially funded by Bayer. Mimi Gao and Wiebke Holkenjans are Bayer employees and may hold shares and/or stock options in the company. Honorine Lardeux, Selin Bagci, Reinhard Pell and Davy Guillarme declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Various single-stranded and hairpin-forming DNA and 2'-O-methyl-RNA oligonucleotides bearing a single (2R,3S)-4-(methoxyamino)butane-1,2,3-triol residue esterified from either O1 and O2 or O1 and O3 were synthesized. Incubation of these oligonucleotides with equimolar mixtures of formylmethyl derivatives of the canonical nucleobases and 2-methylbenzimidazole under mildly acidic conditions revealed base-filling of the modified site to be strongly favored by base stacking of a double-helix, especially an A-type one. In 2'-O-methyl-RNA hairpin oligonucleotides, base-filling of the (2R,3S)-4-(methoxyamino)butane-1,2,3-triol residue with nucleobase aldehydes followed the rules of Watson-Crick base pairing, thymine being the only exception. In single-stranded oligonucleotides or the Hoogsteen strand of triple helices, both the yield and selectivity of base-filling were much more modest., (© 2024 The Author(s). ChemBioChem published by Wiley-VCH GmbH.)

Antisense oligodeoxynucleotides can bind to target RNAs and cleave them using RNase H. Despite the high activity of antisense oligodeoxynucleotides modified with locked nucleic acids (LNA) at several bases at both the 5' and 3' ends (LNA gapmer), toxicity has been reported, necessitating additional backbone modifications to reduce toxicity. In this study, we introduced a sulfonamide linkage into the LNA gapmer to elucidate its fundamental properties such as hybridization, base recognition, and induction of RNase H activity. A new chemically stable sulfonyltriazole was used as a synthetic intermediate to introduce a sulfonamide linkage between the two nucleosides. We studied the properties of the duplex of the sulfonamide-linked gapmer and target RNAs, such as melting temperature, circular dichroism, and cleavage of RNA strands by RNase H. We found that the gapmers had a lower but tolerable duplex stability with base-pair specificity and the ability to induce RNase H activity.

Therapeutic nucleic acids (TNAs) are a new class of drugs that exhibit different properties and mechanisms of action from those of small molecules or biological drugs. Over twenty oligonucleotide drugs and several COVID-19 vaccines have received regulatory approval for clinical use. A characteristic feature of these TNAs is that they are directed against one specific biological target and one specific RNA or DNA sequence. Consequently, TNAs currently used are administered as monotherapy. Due to the known advantages of multidrug therapy with low molecular weight drugs, it may be time to intensify work on such a treatment protocol, also in the case of TNAs., (© 2024 Wiley-VCH GmbH.)

Oligonucleotides (ONs) are an increasingly popular category of molecules in the pharmaceutical landscape, particularly attractive for the treatment of genetic and rare diseases. However, analyzing these molecules presents significant challenges, due to their highly hydrophilic nature, multiple negative charges, and the presence of closely related impurities resulting from the complex solid-phase synthesis process. Ion pairing reverse-phase liquid chromatography (IP-RPLC) is the preferred technique for ONs analysis but is not ideal for mass spectrometry (MS) coupling. Consequently, there is a growing interest in exploring alternative strategies with hydrophilic interaction chromatography (HILIC) emerging as one of the most promising options. As HILIC is not yet fully established for the analysis of ONs, we have prepared this protocol paper to facilitate entry into this field. It not only provides best practices, opportunities, and potential advantages but also caveats and other important considerations for using HILIC to characterize ONs. The paper addresses the selection of stationary and mobile phases, optimization of gradient conditions, MS coupling, and key aspects to consider when manipulating ON samples. We hope this protocol will help establish HILIC as a more universal solution for ONs analysis., (© 2025 Wiley‐VCH GmbH.)

Within the last two decades, the European Medicines Agency and the US Food and Drug Administration have approved several gene therapies. One category is oligonucleotide therapeutics, which allow for the regulation of the expression of target genes. Besides already approved therapeutics, there are several preclinical and clinical trials ongoing. The World Anti-Doping Agency prohibits the use of "nucleic acids or nucleic acid analogs that may alter genome sequences and/or alter gene expression by any mechanism" as a nonspecified method at all times. Hence, the administration of nucleic acids or analogs by athletes would cause an Anti-Doping Rule Violation. Herein, we discuss types of oligonucleotide therapeutics, their potential to be misused in sports, and considerations to sample preparation and mass spectrometric approaches with regard to antidoping analysis., (© 2024 The Author(s). Archiv der Pharmazie published by Wiley‐VCH GmbH on behalf of Deutsche Pharmazeutische Gesellschaft.)

Detection of KRAS mutation in colorectal cancer (CRC) is important in the prediction of response to target therapy. The study aims to develop a novel mutation detection platform called the "PNA-LNA molecular switch" for the detection of KRAS mutation in CRC. We employed the enhanced binding specificity of peptide nucleic acid (PNA) and locked nucleic acid (LNA) in conjunction with a loop-mediated isothermal amplification (LAMP) approach to identify the mutation status of KRAS oncogene codon 12 (c.35G>T/G12V and c.35G>A/G12D) using synthetic oligonucleotides and colon cancer cell lines (Caco-2 and SW480). This method specifically blocked the amplification of the wild-type sequences while substantially amplifying the mutated ones, which was visualized by both colorimetric and fluorescence assays. We then checked the mutation profile of KRAS codon 12 in the DNA derived from tumor tissue samples (number of samples, n = 30) and circulating tumor cells (n = 24) from CRC patients. Finally, we validated the results by comparing them with the data obtained from DNA sequencing of colon tumors (n = 21) of the same CRC patients. This method showed excellent sensitivity (1 DNA copy/μl), reproducibility [relative standard deviation (%RSD) < 5%, for n = 3], and linear dynamic range (1 ag/μl-10 pg/μl, R 2  = 0.94). This platform is significantly faster, relatively cheaper, has superior sensitivity and specificity, and does not require any high-end equipment. To conclude, this method has the potential to be translated into clinical settings for the detection of mutations in diverse diseases and conditions., Competing Interests: Declaration of competing interest The authors declared no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Electrospray mass spectrometry has become indispensable in many disciplines including the classic "omics" techniques such as proteomics or lipidomics, as well as other life science applications in molecular, cellular, and structural biology. However, a limiting factor that often arises for the detection of biomolecular analytes is their poor ionization efficiency in the ion source. Here, we present an add-on device for the electrospray source, termed MS SIEVE (MS Spectral Impurity Eliminator & Value Enhancer), which is placed between the electrospray needle and the cone of the mass spectrometer. We probed the application of MS SIEVE for various biomolecules including proteins, peptides, lipids, glycans and DNA oligonucleotides and even synthetic polymers such as polyethylene glycol and found that MS SIEVE selectively improves the signal intensity, while suppressing the spectral contribution of contaminants such as NaCl. Importantly, MS SIEVE can, in principle, be adapted for any electrospray ion source and, therefore, represents a promising alternative for routine "omics" methods as well as special applications on challenging analytes.

Single-stranded DNA-binding protein (SSB) is essential to DNA replication, DNA repair, and homologous genetic recombination. Our previous study on the crystal structure of a C-terminally truncated SSB from Helicobacter pylori, HpSSBc, in complex with single-stranded DNA (ssDNA) suggests that several aromatic residues, including Phe37, Phe50, Phe56, and Trp84, were involved in ssDNA binding. To investigate the importance of these aromatic residues, the binding activity of four site-directed HpSSB mutants, including F37A HpSSB, F50A HpSSB, F56A HpSSB, and W84A HpSSB, was compared to that of wild-type HpSSB and HpSSBc by means of electrophoresis mobility shift assay (EMSA), tryptophan quenching fluorescence titration, and surface plasmon resonance (SPR). Molecular docking and molecular dynamic (MD) simulation of a F37A and a quadruple mutation model of HpSSBc support that the ssDNA-HpSSBc complex was destabilized when either one or four of the aromatic residues were mutated. The findings of this study suggest that mutation of the phenylalanine and tryptophan residues within the oligonucleotide-binding domain significantly diminished the ssDNA binding capability of HpSSB, highlighting the crucial role these aromatic residues play in the binding of ssDNA by HpSSB., (Copyright © 2024. Published by Elsevier B.V.)