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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.)

Nowadays, nucleic acid derivatives capable of modulating gene expression at the RNA level have gained widespread recognition as promising therapeutic agents. A suitable degree of biological stability of oligonucleotide therapeutics is required for in vivo application; this can be most expeditiously achieved by the chemical modification of the internucleotidic phosphate group, which may also affect their cellular uptake, tissue distribution and pharmacokinetics. Our group has previously developed a strategy for the chemical modification of the phosphate group via the Staudinger reaction on a solid phase of the intermediate dinucleoside phosphite triester and a range of, preferably, electron deficient organic azides such as sulfonyl azides during automated solid-phase DNA synthesis according to the conventional β-cyanoethyl phosphoramidite scheme. Polyfluoro compounds are characterized by unique properties that have prompted their extensive application both in industry and in scientific research. We report herein the synthesis and isolation of novel oligodeoxyribonucleotides incorporating internucleotidic perfluoro-1-octanesulfonyl phosphoramidate or 2,2,2-trifluoroethanesulfonyl phosphoramidate groups. In addition, novel oligonucleotide derivatives with fluorinated zwitterionic phosphate mimics were synthesized by a tandem methodology, which involved ( a ) the introduction of a carboxylic ester group at the internucleotidic position via the Staudinger reaction with methyl 2,2-difluoro-3-azidosulfonylacetate; and ( b ) treatment with an aliphatic diamine, e.g., 1,1-dimethylethylenediamine or 1,3-diaminopropane. It was further shown that the polyfluoro oligonucleotides obtained were able to form complementary duplexes with either DNA or RNA, which were not significantly differing in stability from the natural counterparts. Long-chain perfluoroalkyl oligonucleotides were taken up into cultured human cells in the absence of a transfection agent. It may be concluded that the polyfluoro oligonucleotides described here can represent a useful platform for designing oligonucleotide therapeutics.

New phosphate-modified nucleic acid derivatives are of great significance in basic research and biomedical applications. We have recently developed a new class of phosphoramide benzoazole oligonucleotides (PABAOs). In this work, th properties of N-benzoxazole oligodeoxyribonucleotides have been thoroughly examined. We have demonstrated the convenient automated solid-phase synthesis of oligomers with a different number of modifications (up to six) at various positions. Optical properties, thermal stability, structure, and dynamics of PABAO/DNA complexes have been investigated. The N-benzoxazole-modified phosphate group is uncharged at neutral pH and has a pKa value of 8.52. Structural analysis performed by the CD spectroscopy and MD simulation indicate B-form of PABAO/DNA duplexes. The thermal stability of PABAO/DNA complexes bearing N-benzoxazole is reduced by 2.5-6.2° per modification compared to native duplexes at standard and near physiological buffer conditions. The performed study underlines a great potential of phosphoramide benzoazole oligonucleotides for basic research, applied sciences, and biotechnology., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Alexander A. Lomzov reports a relationship with Russian Science Foundation that includes: funding grants. Alexander A. Lomzov has patent #RU2823099C1 licensed to ICBFM SB RAS. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

We report here the synthesis of tricyclic nucleoside analogues via acid-catalyzed cyclization of guanine with 1,1,3,3-tetramethoxypropane. The method enables the use of hydroxyl-unprotected antiviral drugs (acyclovir, ganciclovir, and penciclovir), guanosines, oligonucleotide, and triazole-linked nucleoside dimers as substrates. Nucleoside trimer and tetramer were synthesized by derivatization reactions.

Antibody-oligonucleotide conjugates (AOCs) are a fast-expanding modality for targeted delivery of therapeutic oligonucleotides to tissues. Here, we present a protocol to generate, purify, and analyze AOCs from off-the-shelf antibodies. We describe steps to conjugate single/double-stranded oligonucleotides bearing amine handles to linkers and, then, to antibodies using well-established chemistry. In addition, we provide details regarding the purification techniques and analytical methods suitable for AOC. This protocol can be applied for several purposes where AOC is a modality of interest. For complete details on the use and execution of this protocol, please refer to Rady et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The clinical translation of oligonucleotide-based therapeutics continues to encounter challenges in delivery. In this study, we introduce a novel class of delivery vehicles for oligonucleotides that are based on poly(ethylene glycol) (PEG) bottlebrush polymers with sequence-defined backbones. Using solid-phase synthesis and bespoke phosphoramidites, the oligonucleotide and the polymer backbone can be assembled on the solid support. The synthesis allows chemical modifiers such as carbon 18 (C 18 ) units to be incorporated into the backbone in specific patterns to modulate the cell-material interactions. Subsequently, PEG side chains were grafted onto the polymer segment of the resulting polymer-oligonucleotide conjugate, yielding bottlebrush polymers. We report an optimal pattern of the C 18 modifier that leads to improved cellular uptake, plasma pharmacokinetics, biodistribution, and antisense activity in vivo . Our results provide valuable insights into the structure-property relationship of polymer-oligonucleotide conjugates and suggest the possibility of tuning the polymer backbone to meet the specific delivery requirements of various diseases.

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.

In this study, we propose a strategy to explore the impact of the proportion of tris(2-carboxyethyl)phosphine (TCEP) and 6-mercaptohexanol (MCH) on the efficiency of oligonucleotide functionalization on PDMS microfluidic channels equipped with pairs of homemade microfabricated platinum microelectrodes. We identified an optimal concentration of these compounds that enables the effective orientation and distribution of probes, thereby facilitating subsequent target hybridization. The experiment included optimizing sample injection into microfluidic channels. We used TCEP as a reducing agent to help the DNA probes adhere to the channel electrode better. This stopped the formation of disulfide bonds during the probe immobilization step. We found the optimal TCEP/MCH mixture ratio (5 mM TCEP and 50 mM MCH), which led to a more uniform distribution and orientation of the DNA probes on the platinum electrode. These optimized conditions resulted in a more compact DNA monolayer and enhanced detection capabilities. The biosensor's performance was evaluated by the detection of the hybridization of complementary DNA sequences in the presence of equimolar Fe(CN) 6 3- /Fe(CN) 6 4- . The detection of the synthetic GP8 resistance gene is facilitated by a measurable decrease in the electron transfer rate, which is directly proportional to its concentration. Under the optimized conditions, the DNA biosensor showed excellent sensitivity (with a detection limit of 10 -17 M) and high specificity when tested against noncomplementary DNA strands.

During the chemical synthesis of the purine riboside, N7-regioisomer is kinetically formed, whereas N9-regioisomer is a thermodynamically formed product. We have studied the effect of substituting N9-regioisomer of guanosine with its N7-regioisomer (N7-guanosine, 7G) at a central position of several RNA duplexes. We found that this single substitution by 7G severely diminished their thermodynamic stabilities when 7G paired with C and U, but remarkably, led to a significant amount of stabilization in most of the duplexes when forming mismatches with G and A. The extent of stabilization was observed to be dependent on the sequence and orientation of neighboring base pairs of N7-guanosine. 1D and 2D NMR studies on the duplexes along with extensive molecular dynamics simulations revealed the conformational differences occurring due to the substitution of G by 7G, and it was observed that the thermodynamic results were largely explainable by considering the formation of stable noncanonical hydrogen bonding interactions, although other interactions such as stacking and electrostatic interactions could also play a role. These observations can have important applications in the design of RNA-based disease diagnostics and therapeutics., (© 2025 Jarmolowicz et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

The use of liquid chromatography coupled with mass spectrometry (LC-MS) for the characterization of oligonucleotides and nucleic acids is a powerful analytical method. Recently, hydrophilic interaction chromatography (HILIC) has been proposed as a reasonable alternative to ion-pair reversed phase separations of oligonucleotides prior to MS. A wide variety of HILIC stationary phase surface chemistries are currently available. Although their selectivity can be considerably different, few studies have compared these chemistries for LC-MS analysis of oligonucleotides. We evaluated ten different HILIC column chemistries to understand their capabilities for separating a variety of oligonucleotides. In general, we found that most columns were ineffective at separating larger ( n > 15-mer) oligonucleotides under the mobile phase and gradient conditions evaluated here. However, several stationary phases were found to be effective for separating smaller oligonucleotides such as endonuclease digestion products. Given that early eluting oligonucleotides were found to be compatible with standard electrospray ionization conditions, several different HILIC stationary phase options are available for LC-MS studies of smaller oligonucleotides including those generated in RNA modification mapping experiments.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death, but effective therapeutic strategies are limited. Transcriptional factor c-Myc plays an oncogenic role in tumorigenesis and is an attractive target for HCC treatment. However, targeted therapy against c-Myc remains challenging. Herein, by conjugating VH032 with an optimized DNA sequence that recognized c-Myc complex, we discovered oligonucleotide-based proteolysis targeting chimeras (PROTACs), termed as MP-16 and MP-17, which effectively induced degradation of c-Myc. Mechanically, MP-16 or MP-17 directly interacted with c-Myc complex to form VHL/PROTAC/c-Myc ternary complex, and triggered c-Myc degradation by recruiting ubiquitin-proteasome system, suppressing cell proliferation of HCC cells. In mice model, MP-16 or MP-17 significantly inhibited HCC tumor growth and exhibited promising drug safety. This work provided novel oligonucleotide PROTACs that degraded c-Myc, giving a new lead structure for HCC therapy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

The development of new convenient tools for the design of multicomponent nucleic acid (NA) complexes is one of the challenges in biomedicine and NA nanotechnology. In this paper, we analyzed the formation of hybrid RNA/DNA concatemers and self-limited complexes by a pair of oligonucleotides using UV melting, circular dichroism spectroscopy, and a gel shift assay. Effects of the size of the linker between duplex-forming segments of the oligonucleotides on complexes' shape and number of subunits were compared and systematized for RNA/DNA, DNA/DNA, and RNA/RNA assemblies. The data on complex types summarized here as heat maps offer a convenient tool for the design of NA constructs. General rules found for RNA/DNA, DNA/DNA, and RNA/RNA complexes allow not only designing complexes with desired structures but also purposefully transforming their geometry. The A-form of the double helix of the studied RNA/DNA complexes was confirmed by circular dichroism analysis. Moreover, we show for the first time efficient degradation of RNA in hybrid self-limited complexes by RNase H and imidazole. The results open up new prospects for the design of supramolecular complexes as tools for nanotechnology, nanomachinery, and biomedical applications.

Carbon dots (CDs) represent an emerging class of nanomaterials that combine outstanding photoluminescent properties with low toxicity and excellent biocompatibility. These unique features have garnered significant interest for potential applications in sensing as well as nanovectors for bioactive compounds. Within this context, the possibility of synthesizing chiral carbon dots (CCDs) has paved the way for a plethora of bioapplications in their interaction with chiral biomolecules. In this study we report the synthesis and characterization of CCDs with opposite chiralities and their selective interaction with nucleic acids. A systematic study on their interaction with different oligonucleotides (ODNs) using UV-vis, photoluminescence, and circular dichroism analyses highlighted how the chiral surface of the CCDs induces distinct spectroscopic responses in CCDs-ODN conjugates. These findings establish the foundation for innovative applications of CCDs as nanosensors and nanocarriers for nucleic acids. Additionally, the antioxidant properties of CCDs were investigated, highlighting their dual potential as both sensing and preservative nanomaterials for genetic material. Our results suggest significant implications for the development of chiral-specific diagnostic tools, drug delivery systems, and therapeutic agents. Furthermore, these properties open new avenues for the use of CCDs in antibiotic residue detection, fluorescence imaging, and photodynamic therapy., (© 2024 The Author(s). Chemistry - A European Journal published by Wiley-VCH GmbH.)

Deposits of aggregated TAR DNA-binding protein 43 (TDP-43) in the brain are associated with several neurodegenerative diseases. It is well established that binding of RNA/DNA to TDP-43 can prevent TDP-43 aggregation, but an understanding of the structure(s) and conformational dynamics of TDP-43, and TDP-43-RNA complexes, is lacking, including knowledge of how the solution environment modulates these properties. Here, we address this challenge using hydrogen-deuterium exchange-mass spectrometry. In the presence of RNA olignoucleotides, we observe protection from exchange in the RNA recognition motif (RRM) domains of TDP-43 and the linker region between the RRM domains, consistent with nucleic acid binding modulating interdomain interactions. Intriguingly, at elevated salt concentrations, the extent of protection from exchange is reduced in the RRM domains when bound to an RNA sequence derived from the 3' UTR of the TDP-43 mRNA (CLIP34NT) compared to when bound to a (UG)6 repeat sequence. Under these conditions, CLIP34NT is no longer able to prevent TDP-43 aggregation. This suggests that a salt-induced structural rearrangement occurs when bound to this RNA, which may play a role in facilitating aggregation. Additionally, upon RNA binding, we identify differences in exchange within the short α-helical region located in the C-terminal domain (CTD) of TDP-43. These allosterically altered regions may influence the ability of TDP-43 to aggregate and fine-tune its RNA binding repertoire. Combined, these data provide additional insights into the intricate interplay between TDP-43 aggregation and RNA binding, an understanding of which is crucial for unraveling the molecular mechanisms underlying TDP-43-associated neurodegeneration.

The RNA World hypothesis posits that RNA was the molecule of both heredity and function during the emergence of life. This hypothesis implies that RNA templates can be copied, and ultimately replicated, without the catalytic aid of evolved enzymes. A major problem with nonenzymatic template-directed polymerization has been the very poor copying of sequences containing rA and rU. Here, we overcome that problem by using a prebiotically plausible mixture of RNA mononucleotides and random-sequence oligonucleotides, all activated by methyl isocyanide chemistry, that direct the uniform copying of arbitrary-sequence templates, including those harboring rA and rU. We further show that the use of this mixture in copying reactions suppresses copying errors while also generating a more uniform distribution of mismatches than observed for simpler systems. We find that oligonucleotide competition for template binding sites, oligonucleotide ligation and the template binding properties of reactant intermediates work together to reduce product sequence bias and errors. Finally, we show that iterative cycling of templated polymerization and activation chemistry improves the yields of random-sequence products. These results for random-sequence template copying are a significant advance in the pursuit of nonenzymatic RNA replication., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Nucleic acids, with their unique duplex structure, which is key for information replication, have sparked interest in self-replication's role in life's origins. Early template-based replicators, initially built on short oligonucleotides, expanded to include peptides and synthetic molecules. We explore here the potential of a class of synthetic duplex-forming oligoanilines, as self-replicators. We have recently developed oligoanilines equipped with 2-trifluoromethylphenol-phosphine oxide H-bond base pairs and we investigate whether the imine formed between aniline and aldehyde complementary monomers can self-replicate. Despite lacking a clear sigmoidal kinetic profile, control experiments with a methylated donor and a competitive inhibitor support self-replication. Further investigations with the reduced aniline dimer demonstrate templated synthesis, revealing a characteristic parabolic growth. After showing sequence selective duplex formation, templated synthesis and the emergence of catalytic function, the self-replication behaviour further suggests that the unique properties of nucleic acids can be paralleled by synthetic recognition-encoded molecules., (© 2024 The Author(s). Chemistry - A European Journal published by Wiley-VCH GmbH.)

2',4'-methylene bridged nucleic acid/locked nucleic acid (2',4'-BNA/LNA; LNA) is a modified nucleic acid that improves the function of antisense oligonucleotide therapeutics. In particular, LNA in the DNA strand increases its binding affinity for the target RNA. Predicting the binding affinities of LNA-containing antisense oligonucleotides and RNA duplexes is useful for designing antisense oligonucleotides. The nearest neighbor parameters may be useful for binding affinity prediction, similar to those for natural nucleic acids. However, the sequence dependence of the thermodynamic stability of DNA/RNA duplexes containing LNA remains unexplored. Therefore, in this study, we evaluated the thermodynamic stabilities of DNA/RNA duplexes containing a single LNA modification in the DNA strand. We found that LNA-stabilized DNA/RNA duplexes averaged -1.5 kcal mol -1 . Our findings suggest that the thermodynamic stabilization effect of LNA is sequence-specific.

Globally, widespread tuberculosis is one of the acute problems of healthcare. Drug-resistant forms of tuberculosis require a personalized approach to treatment. Currently, rapid methods for detecting drug resistance of Mycobacterium tuberculosis (MTB) to some antituberculosis drugs are often used and involve optical, electrochemical, or PCR-based assays. Despite the large number of these assays, it is necessary to develop new tests (for drug-resistant MTB strains) that are structurally simple and do not require specialized equipment. Colorimetric assays involving a colloidal solution of gold nanoparticles (AuNPs) have good potential for the development of the needed diagnostic tools. Here, conditions were found for the formation of tandem duplexes between DNA probes and DNA targets, representing a part of MTB gene gyrA , either wildtype or containing a single-nucleotide polymorphism associated with fluoroquinolone resistance of MTB. Adsorption of the duplexes on AuNPs allowed to distinguish the two targets owing to the formation of nano-constructs of different structures. Interaction of DNA with AuNPs was analyzed by optical spectroscopy, dynamic light scattering, and transmission electron microscopy. A scheme is proposed for direct colorimetric detection of the fluoroquinolone-resistance-associated single-nucleotide polymorphism at a 2 nM concentration in a liquid system based on a shift of AuNPs' optical absorption maximum.

DNA photo-crosslinking reactions occur widely in biological systems and are often used as valuable tools in molecular biology. In this article, we demonstrate the application of an oligonucleotide 5-fluoro-2'-O-methyl-4-thiouridine ( FS U )-containing probe for the fluorescent detection of specific DNA sequences. The design of the probe was predicated on studies of agents that could adversely affect its efficiency. The most important of these is the intrastrand photo-crosslinking of single-stranded oligodeoxynucleotides bearing FS U . Our research findings indicate that FS U after photoexcitation can react with nonadjacent bases; specifically, it can react with distant thymine and cytosine residues in the chain, forming fluorescent and nonfluorescent intrastrand crosslinks, respectively. In addition, partial photooxidation of the FS U residue to 5-fluorouridine was also observed. The results of the study are significant in terms of the use of FS U -labeled oligonucleotide probes in the fluorescence-based detection of specific DNA sequences because the creation of a fluorescent intrastrand crosslink could produce a false signal. To overcome this problem, replacing thymidine with deoxyuridine in the FS U -labeled oligonucleotide probe is proposed and tested.

The polar nature of nucleobases, nucleosides and nucleotides makes hydrophilic interaction chromatography (HILIC) a good choice of technology for separation. Both naturally occurring and modified nucleosides and nucleotides have been successfully separated in HILIC. A wide range of stationary phases with different retention and selectivity are suitable for the separation of nucleobases, nucleosides and nucleotides; and a sufficient knowledge base is also available to guide method development. Although oligonucleotides are significantly different from nucleotides in terms of polarity and charges, HILIC has been shown to be a viable alternative to ion-pairing reversed-phase liquid chromatography (IP-RPLC). Only a few polar stationary phases have been shown to provide satisfactory performance; however, the requirements for the mobile phase composition including organic solvent, mobile phase pH and salt concentration are sufficiently understood. This review provides a comprehensive evaluation of the chromatographic conditions with a historical perspective on adopting and developing HILIC for the separation of nucleobases, nucleosides, nucleotides and oligonucleotides. The areas for more research and potential directions for future development activities are identified and discussed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Phosphorodiamidate Morpholino Oligonucleotides (PMOs) have been well established in the milieu of FDA-approved oligonucleotide-based drugs in the past decade. Given their relevance in antisense therapeutics, a DNA/RNA synthesizer-compatible modular synthesis protocol of PMOs is long awaited to explore next-generation PMO chimeras with other therapeutically proven oligonucleotide backbones. Herein, we demonstrate a streamlined 5' → 3'phosphoramidite approach for the synthesis of PMOs using tert -butyl-protected 5'- t Bu- morpholino phosphoramidites, which were synthesized from 5'-OH morpholino monomers derived from commercially available ribonucleosides. 2-Cyanoethyl (CE)-protected 5'-CE-morpholino phosphoramidites were also synthesized to generate thiophosphoramidate (TMO) and phosphoramidate (MO) morpholino oligonucleotides. Full-length PMOs and TMOs in exceptional overall yields were obtained with operational simplicity and compatibility with automated DNA/RNA synthesizers utilizing controlled pore glass (CPG) as the solid support and CH 3 CN as the solvent. Importantly, this method has opened the possibility of designing various biologically relevant ASO design modalities, such as PMO-TMO and PMO-MO, which were inaccessible otherwise. Moreover, DNA nucleotides were also incorporated to generate PMO-psDNA and PMO-DNA using commercially available 5'-DNA phosphoramidites. The biophysical properties of all synthesized oligonucleotides were analyzed using UV melting and circular dichroism studies. The serum interaction profile and innate immune response of the PS-modified polythymidine oligonucleotides were analyzed and it was found that the mixed backbone oligonucleotides had a balanced profile compared to fully charged or neutral extremities. Overall, the synthesis is amenable to fast generation of various types of PMO chimeras for biological screening, which will expedite their therapeutic exploration and transition to clinic.

Antisense oligonucleotide (ASO) therapies hold significant promise in the realm of molecular medicine. By precisely targeting RNA molecules, ASOs offer an approach to modulate gene expression and protein production, making them valuable tools for treating a wide range of genetic and acquired diseases. As the precise intracellular targeting and delivery of ASOs is challenging, strategies for preparing ASO-ligand conjugates are in exceedingly high demand. This work leverages the utility of native chemical ligation to conjugate ASOs with therapeutically relevant chemical modifications including locked nucleic acids and phosphorothioate backbone modifications to peptides and sugars via a stable amide linkage. A suite of post-ligation functionalizations through modification of the cysteine ligation handle are highlighted, including chemoselective radical desulfurization, lipidation, and alkylation with a range of valuable handles (e.g. alkyne, biotin, and radionuclide chelating ligands), affording multifunctional constructs for further applications in biology and medicine. Application of the methodology to a clinically-relevant triantennary-GalNAc ASO conjugate and validation of its binding and functional activity underpins the applicability of the technique to oligonucleotide-based therapeutics., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

The study of the interactions between biofunctionalized gold nanoclusters (Au NCs) and spermatozoa is highly relevant to evaluate the potential of Au NCs as imaging probes and transfection agents in reproductive biology. In this work, confocal laser scanning microscopy (CLSM) was used to investigate the distribution of Au NCs bioconjugated with peptide (nuclear localisation sequence, NLS) and oligonucleotide (locked nucleic acid, LNA) ligands in bovine spermatozoa. Fluorescence lifetime imaging (FLIM) was employed to detect changes in the NC's chemical environment. We observed a pronounced regio-selective accumulation of the bioconjugates in spermatozoa with high concentration at the equatorial segment. Furthermore, 3D-CLSM showed successful non-endosomal cellular uptake of the conjugates by intact sperm cells and the distribution of the bioconjugates was found to be influenced by the ligand types. Interestingly, the FLIM data showed differences in lifetime depending on membrane integrity. Furthermore, ligand-dependent changes in lifetime between NC bioconjugates carrying peptide and oligonucleotide ligands were found, probably attributed to specific interactions with sperm cell compartments., (© 2024 The Author(s). ChemBioChem published by Wiley-VCH GmbH.)

The Watson-Crick base pairing property of DNA is widely used for fabricating DNA nanostructures with well-defined geometry. Moreover, DNA nanostructures can be easily modified in terms of shape, size and function at the nanoscale level. Therefore, investigation on smaller and stable branched DNA (bDNA) is of critical significance for biomedical applications. In the present communication, we report smaller and stable branched DNA (bDNA) which is of critical significance for biomedical applications. In this study, a novel strategy has been used in identifying stable bDNA nanostructures with a minimum number of Watson-Crick base pairings. The importance of hybridizing regions and helical twists between multiple oligonucleotides has been explored using various biophysical techniques. The electrophoretic analysis demonstrated that hybridizing regions with ≥12 nt nucleotides can form stable bDNA structures. Substantial negative enthalpic contributions determine the significance of base stacking and the length of oligonucleotides in the hybridization process. Finally, thermal melting investigations confirmed the creation of bDNA nanostructures with ≥12 nt long hybridizing regions. In general, our findings indicate that bDNA oligonucleotides do not undergo hybridization if the number of base pairs is lesser for a single helical turn. Furthermore, the yield and stability of smaller bDNA nanostructures in physiological conditions are comparable with the earlier reported higher-order structures. Hence, smaller bDNAs are more stable which may be preferred over conventional bDNA nanostructures for advanced biomedical applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

An effective delivery platform is crucial for the development of mRNA vaccines and therapeutics. Here, a versatile platform utilizing cholesterol-modified oligonucleotides (L-oligo) that bind to the mRNA within lipid nanoparticles (LNP), and enables the effective delivery of the mRNA into target cells is introduced. mRNA incorporated into LNPs via linkage with L-oligo, termed oligonucleotide-linked LNP (lnLNP), is superior in cellular uptake and transfection efficiency in target cells in vitro and in vivo, compared to the conventional LNP formulations. It is further applied lnLNP as an mRNA vaccine platform for SARS-CoV-2, demonstrating robust induction of neutralizing activity as well as polyfunctional SARS-CoV-2-specific T-cell response in vivo. The current strategy can be versatilely applied to different LNP platforms, for vaccine and therapeutic applications against various diseases, such as infections and cancers., (© 2024 Wiley‐VCH GmbH.)

With recent advancements concerning the optimization of the analytical conditions, it is feasible to analyze polar molecules using supercritical fluid chromatography (SFC). In this study, the applicability of SFC is evaluated for analyzing 5-, 10-, 15-, and 18-mer oligonucleotides, and SFC is then applied to analyze deaminated products, which are side products generated during oligonucleotide synthesis. These side products are difficult to separate from the target oligonucleotide, with the difficulty varying depending on the deamination position and sequences, even when using ion-pair reversed-phase liquid chromatography (IP-RPLC), a common method for oligonucleotide analysis. Our results demonstrate that SFC, with octylamine as a modifier additive, can achieve sharp chromatographic peaks for 5-, 10-, 15-, and 18-mer oligonucleotides modified with 2'-O-methoxyethyl RNA (2'-MOE), regardless of the presence of the hydrophobic 4,4'-dimethoxytrityl (DMTr) group on the sequence. After optimization of the column oven temperature, modifier additive, and stationary phase, SFC successfully separated oligonucleotides with various numbers and positions of deamination from the target oligonucleotide. SFC exhibited different selectivities for DMTr-on and DMTr-off oligonucleotides compared with those for IP-RPLC, which indicates that SFC can serve as a valuable alternative tool for the purification and analysis of oligonucleotides., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025. Published by Elsevier B.V.)