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Since its inception, antisense oligonucleotides (ASOs) have been extensively used in functional genomics. Recent advancements in chemical modification and delivery technology extend its applications to the targeted management of crop pests. ASO was conceptualized in the early 1970s and first applied to inhibit retrovirus replication in chickens. The principles of ASOs rely on target complementarity by hybridizing with the mRNA and inhibiting the function. However, the application of ASO received little attention because of its high instability in the biological environment. Initial ASOs, featuring phosphorothioate linkages, laid the groundwork for subsequent advancements, by addressing challenges such as nuclease degradation and cellular uptake limitations. The second generation introduced 2'-modifications to enhance stability and specificity while allowing reversible gene regulation. Third-generation ASOs incorporated innovative chemical modifications like locked nucleic acids (LNAs), peptide nucleic acids (PNAs), phosphoramidate morpholino oligomers (PMOs), 2′-deoxy-2′-fluoro-d-arabinonucleic acid (FANA), cyclohexene nucleic acids (CeNAs), and tricyclo-DNA (tcDNA), offering enhanced efficacy and stability. ASO technology continues to evolve, promising precision medicine and customized therapies for genetic diseases. In agriculture, ASOs selectively target essential genes in pests or pathogens, which would be a novel option for providing precise crop protection and reducing reliance on conventional pesticides. Recent studies demonstrated successful ASO application in thrips, psyllids, Gypsy moths, scale insects, citrus greening, and Zebra-chip diseases. Additionally, ASOs hold the potential for improving crop yield and quality by modulating gene expression related to plant growth, development, and stress responses. They can enhance abiotic stress tolerance, regulate genes associated with specific traits, and offer alternative strategies for managing diseases, such as targeting S-genes in tomatoes for resistance against bacterial spots. These applications antecede the oligo-based pesticides that will revolutionize future agriculture by reducing pesticide uses and harmful impacts on the ecosystem. Further advancements in environmental stability, field efficacy, delivery formulation, and environmental safety would mold this technology into a novel tool for sustainable crop protection. This review highlights the fundamentals of ASO, recent advancements in design chemistry, delivery systems, validation and optimization process, and its potential in customized pest management. This would serve as a ready reckoner for future ASO research and application. [ABSTRACT FROM AUTHOR]

Locked nucleic acids (LNAs) are a subtype of antisense oligonucleotides (ASOs) that are characterized by a bridge within the sugar moiety. LNAs owe their robustness to this chemical modification, which as the name suggests, locks it in one conformation. This perspective includes two components: a general overview on ASOs from one side and on delivery issues focusing on lipid nanoparticles (LNPs) on the other side. Throughout, a screening of the ongoing clinical trials involving ASOs is given, as well as a take on the versatility and challenges of using LNAs. Finally, we highlight the potential of LNPs as carriers for the successful delivery of LNAs.

2',4'-Bridged modifications such as 2'-O,4'-C-methylene-bridged nucleotides (LNAs) and 2'-O,4'-C-ethylene-bridged nucleotides (ENAs) provide high binding affinity for duplex formation. Stabilization by the introduction of the bridged nucleic acids is considered to be due to pre-organization. In this study, we found that the introduction of 2',4'-C-bridged 2'-deoxynucleotides (CRNs; Conformationally Restricted Nucleotides) into DNA/RNA duplexes leads to destabilization, as opposed to the previously accepted notion that 2',4'-bridged modifications always lead to stabilization. [ABSTRACT FROM AUTHOR]

Ribosomal frameshifting (RFS) at the slippery site of SARS-CoV-2 RNA is essential for the biosynthesis of the viral replication machinery. It requires the formation of a pseudoknot (PK) structure near the slippery site and can be inhibited by PK-disrupting oligonucleotide-based antivirals. We obtained and compared three types of such antiviral candidates, namely locked nucleic acids (LNA), LNA–DNA gapmers, and G-clamp-containing phosphorothioates (CPSs) complementary to PK stems. Using optical and electrophoretic methods, we showed that stem 2-targeting oligonucleotide analogs induced PK unfolding at nanomolar concentrations, and this effect was particularly pronounced in the case of LNA. For the leading PK-unfolding LNA and CPS oligonucleotide analogs, we also demonstrated dose-dependent RSF inhibition in dual luciferase assays (DLAs). Finally, we showed that the leading oligonucleotide analogs reduced SARS-CoV-2 replication at subtoxic concentrations in the nanomolar range in two human cell lines. Our findings highlight the promise of PK targeting, illustrate the advantages and limitations of various types of DNA modifications and may promote the future development of oligonucleotide-based antivirals. [ABSTRACT FROM AUTHOR]

Abstract Originally discovered by Nielsen in 1991, peptide nucleic acids and other artificial genetic polymers have gained a lot of interest from the scientific community. Due to their unique biophysical features these artificial hybrid polymers are now being employed in various areas of theranostics (therapy and diagnostics). The current review provides an overview of their structure, principles of rational design, and biophysical features as well as highlights the areas of their successful implementation in biology and biomedicine. Finally, the review discusses the areas of improvement that would allow their use as a new class of therapeutics in the future.

The introduction of sulfur into the phosphate linkage of chemically synthesized oligonucleotides creates the stereocenters on phosphorus atoms. Researchers have valued the nature of backbone stereochemistry and early on investigated drug properties for the individual stereocenters in dimers or short oligomers. Only very recently, it has become possible to synthesize fully stereodefined antisense oligonucleotides in good yield and purity. Non-bridging phosphorodithioate (PS2) introduces second sulfur into the phosphorothioate linkage to remove the chirality of phosphorus atom. Here, we describe the application of symmetrical non-bridging PS2 linkages in the context of stereodefined locked nucleic acids (LNAs) antisense oligonucleotides with the goal of reducing chiral complexity and, ultimately, resulting in single molecules. In addition, we propose a rather simple strategy to rapidly identify stereodefined gapmers, combining PS2 and a preferred stereochemistry motif (RSSR), which supports RNase-H-mediated target knockdown. Pharmacological efficacy and metabolic stability are investigated systematically using ApoB as a target sequence, where in vivo data correlate well to what is observed in vitro.

Salmonella enteritidis (SE) is an important factor causing foodborne disease, and electrochemical sensors have drawn much attention for SE prevention and detection due to their many advantages. A renewable electrochemical sensor using specially designed locked nucleic acids (LNA) as linkers for the detection of SE was proposed to improve the reusability and reproducibility of biosensors. One end of the LNA was designed as an anchor to attach to modified electrodes through the sulfhydryl group; the other end was used to match with a short segment of SE aptamers, which will allow for the convenient renewal of occupied aptamers by raising the temperature. Results revealed that the manufactured biosensor had good stability, reproducibility, and selectivity in addition to a linear range of 6 × 101–6 × 105 CFU/mL and a limit of detection (LOD) of 20.704 CFU/mL. The recovery rate of SE for the real sample varied from 98.84% to 134.82% without exceeding 16.27% in the relative standard deviation (RSD). The proposed biosensor appears to be a promising tool for foodborne pathogen detection. [ABSTRACT FROM AUTHOR]

Originally discovered by Nielsen in 1991, peptide nucleic acids and other artificial genetic polymers have gained a lot of interest from the scientific community. Due to their unique biophysical features these artificial hybrid polymers are now being employed in various areas of theranostics (therapy and diagnostics). The current review provides an overview of their structure, principles of rational design, and biophysical features as well as highlights the areas of their successful implementation in biology and biomedicine. Finally, the review discusses the areas of improvement that would allow their use as a new class of therapeutics in the future. [ABSTRACT FROM AUTHOR]

Chemically modified oligonucleotides can solve biosensing issues for the development of capture probes, antisense, CRISPR/Cas, and siRNA, by enhancing their duplex-forming ability, their stability against enzymatic degradation, and their specificity for targets with high sequence similarity as microRNA families. However, the use of modified oligonucleotides such as locked nucleic acids (LNA) for biosensors is still limited by hurdles in design and from performances on the material interface. Here we developed a fluorogenic biosensor for non-coding RNAs, represented by polymeric PEG microgels conjugated with molecular beacons (MB) modified with locked nucleic acids (MicroLOCK). By 3D modeling and computational analysis, we designed molecular beacons (MB) inserting spot-on LNAs for high specificity among targets with high sequence similarity (95%). MicroLOCK can reversibly detect microRNA targets in a tiny amount of biological sample (2 μL) at 25 °C with a higher sensitivity (LOD 1.3 fM) without any reverse transcription or amplification. MicroLOCK can hybridize the target with fast kinetic (about 30 min), high duplex stability without interferences from the polymer interface, showing high signal-to-noise ratio (up to S/N = 7.3). MicroLOCK also demonstrated excellent resistance to highly nuclease-rich environments, in real samples. These findings represent a great breakthrough for using the LNA in developing low-cost biosensing approaches and can be applied not only for nucleic acids and protein detection but also for real-time imaging and quantitative assessment of gene targeting both in vitro and in vivo. [Display omitted] [ABSTRACT FROM AUTHOR]

Ribosomal frameshifting (RFS) at the slippery site of SARS-CoV-2 RNA is essential for the biosynthesis of the viral replication machinery. It requires the formation of a pseudoknot (PK) structure near the slippery site and can be inhibited by PK-disrupting oligonucleotide-based antivirals. We obtained and compared three types of such antiviral candidates, namely locked nucleic acids (LNA), LNA–DNA gapmers, and G-clamp-containing phosphorothioates (CPSs) complementary to PK stems. Using optical and electrophoretic methods, we showed that stem 2-targeting oligonucleotide analogs induced PK unfolding at nanomolar concentrations, and this effect was particularly pronounced in the case of LNA. For the leading PK-unfolding LNA and CPS oligonucleotide analogs, we also demonstrated dose-dependent RSF inhibition in dual luciferase assays (DLAs). Finally, we showed that the leading oligonucleotide analogs reduced SARS-CoV-2 replication at subtoxic concentrations in the nanomolar range in two human cell lines. Our findings highlight the promise of PK targeting, illustrate the advantages and limitations of various types of DNA modifications and may promote the future development of oligonucleotide-based antivirals.

Controlled enzymatic synthesis is an alluring alternative to solid‐phase synthesis and polymerase‐mediated incorporation of nucleotides for the crafting of chemically modified, therapeutic oligonucleotides. While this approach has met some success for the elaboration of long, unmodified DNA sequences, very little research efforts have been dedicated to xeno nucleic acids (XNAs). Here, we have evaluated the possibility of using various 3'‐O‐blocking groups for controlled synthesis of locked nucleic acids (LNA). LNA nucleosides were equipped with protecting groups used in synthetic organic chemistry and were evaluated for their stability. The most promising candidates, benzoyl‐ and pivaloyl‐protected nucleosides, were converted to the corresponding nucleotides. The resulting modified nucleotides were shown to be accepted by various polymerases. While single incorporation events were observed in high yields, strong esterase activity of polymerases represents a lasting hurdle that needs to be overcome. Overall, this article represents an additional step towards the controlled enzymatic synthesis of LNA‐containing oligonucleotides and could be extended to other sugar or nucleobase modified nucleotides. [ABSTRACT FROM AUTHOR]

A large number of Locked Nucleic Acids (LNAs) with variety of modifications and restricted conformations have been developed in the last few decades. These modifications have significantly improved the biological properties of oligonucleotides, when LNAs moieties were incorporated into them. Herein, the synthesis and applications of these modified locked nucleic acids as antisense oligonucleotides are discussed. Supplemental data for this article is available online at https://doi.org/10.1080/15257770.2022.2052316. [ABSTRACT FROM AUTHOR]

Salmonella enteritidis (SE) is an important factor causing foodborne disease, and electrochemical sensors have drawn much attention for SE prevention and detection due to their many advantages. A renewable electrochemical sensor using specially designed locked nucleic acids (LNA) as linkers for the detection of SE was proposed to improve the reusability and reproducibility of biosensors. One end of the LNA was designed as an anchor to attach to modified electrodes through the sulfhydryl group; the other end was used to match with a short segment of SE aptamers, which will allow for the convenient renewal of occupied aptamers by raising the temperature. Results revealed that the manufactured biosensor had good stability, reproducibility, and selectivity in addition to a linear range of 6 × 101–6 × 105 CFU/mL and a limit of detection (LOD) of 20.704 CFU/mL. The recovery rate of SE for the real sample varied from 98.84% to 134.82% without exceeding 16.27% in the relative standard deviation (RSD). The proposed biosensor appears to be a promising tool for foodborne pathogen detection.

The photothermal effect of plasmonic nanostructures has numerous applications, such as cancer therapy, photonic gene circuit, large cargo delivery, and nanostructure-enhanced laser tweezers. The photothermal operation can also induce unwanted physical and biochemical effects, which potentially alter the cell behaviors. However, there is a lack of techniques for characterizing the dynamic cell responses near the site of photothermal operation with high spatiotemporal resolution. In this work, we show that the incorporation of locked nucleic acid probes with gold nanorods allows photothermal manipulation and real-time monitoring of gene expression near the area of irradiation in living cells and animal tissues. The multimodal gold nanorod serves as an endocytic delivery reagent to transport the probes into the cells, a fluorescence quencher and a binding competitor to detect intracellular mRNA, and a plasmonic photothermal transducer to induce cell ablation. We demonstrate the ability of the gold nanorod-locked nucleic acid complex for detecting the spatiotemporal gene expression in viable cells and tissues and inducing photothermal ablation of single cells. Using the gold nanorod-locked nucleic acid complex, we systematically characterize the dynamic cellular heat shock responses near the site of photothermal operation. The gold nanorod-locked nucleic acid complex enables mapping of intracellular gene expressions and analyzes the photothermal effects of nanostructures toward various biomedical applications.

Locked nucleic acids (LNAs) are a subtype of antisense oligonucleotides (ASOs) that are characterized by a bridge within the sugar moiety. LNAs owe their robustness to this chemical modification, which as the name suggests, locks it in one conformation. This perspective includes two components: a general overview on ASOs from one side and on delivery issues focusing on lipid nanoparticles (LNPs) on the other side. Throughout, a screening of the ongoing clinical trials involving ASOs is given, as well as a take on the versatility and challenges of using LNAs. Finally, we highlight the potential of LNPs as carriers for the successful delivery of LNAs., Competing Interests: D.P. receives licensing fees (to patents on which he was an inventor) from, invested in, consults (or on scientific advisory boards or boards of directors) for, lectured (and received a fee), or conducts sponsored research at TAU for the following entities: ART Biosciences, BioNtech SE, Earli Inc., Kernal Biologics, Geneditor Biologics, Newphase Ltd., NeoVac Ltd., Roche, SirTLabs Corporation, and Teva Pharmaceuticals Inc., (© 2024 The Author(s).)

Nucleosides and their analogues have been in clinical use since 1950s, and particularly of guanine that have become cornerstone for the treatment of cancer and viral infections. But this rejoice of therapeutic potential is hampered by the intricate glycosylation of guanine, which usually results in a mixture ofN-7 and V-9 guanine nucleosides. The present review is focused on the medicinal importance and different strategies undertaken for regioselective synthesis of guanine nucleosides. [ABSTRACT FROM AUTHOR]

Background/Aim: Multiple myeloma is a B-cell neoplasm, which can spread within the marrow of the bones forming many small tumors. In advanced disease, multiple myeloma can spread to the blood as plasma cell leukemia. In some cases, a localized tumor known as plasmacytoma is found within a single bone. Despite the approval of several agents such as melphalan, corticosteroids, proteasome inhibitors, thalidomide-based immuno-modulatory agents, histone deacetylase inhibitors, a nuclear export inhibitor and monoclonal antibodies daratuzumab and elatuzumab, the disease presently remains uncurable. Materials and Methods: In order to define new targets and treatment modalities we searched the literature for microRNAs, which increase or inhibit in vivo efficacy in multiple-myelomarelated xenograft models. Results and Conclusion: We identified six up-regulated and twelve down-regulated miRs, which deserve further preclinical validation. [ABSTRACT FROM AUTHOR]

Aim: Oligonucleotide therapeutics can be quantified using various bioanalytical methods, and these methods have been compared extensively. However, few comparisons exist where the same analyte is evaluated by multiple assay platforms. Materials & methods: Hybrid LC-MS, SPE-LC-MS, HELISA and SL-RT-qPCR methods were developed for an siRNA analyte, and samples from a pharmacokinetic study were analyzed by all four methods. Results: All assay platforms provided comparable data, though higher concentrations were observed using the non-LC-MS assays. Hybrid LC-MS and SL-RT-qPCR were the most sensitive methodologies, and SL-RT-qPCR and HELISA demonstrated the highest throughput. Conclusion: Each assay platform is suitable for oligonucleotide bioanalysis, and the ultimate choice of methodology will depend on the prioritization of needs such as sensitivity, specificity and throughput.

2',4'-Bridged modifications such as 2'- O ,4'- C -methylene-bridged nucleotides (LNAs) and 2'- O ,4'- C -ethylene-bridged nucleotides (ENAs) provide high binding affinity for duplex formation. Stabilization by the introduction of the bridged nucleic acids is considered to be due to pre-organization. In this study, we found that the introduction of 2',4'- C -bridged 2'-deoxynucleotides (CRNs; Conformationally Restricted Nucleotides) into DNA/RNA duplexes leads to destabilization, as opposed to the previously accepted notion that 2',4'-bridged modifications always lead to stabilization.

DNA and locked nucleic acid (LNA) were characterized as single strands, as well as double stranded DNA-DNA duplexes and DNA-LNA hybrids using tandem mass spectrometry with collision-induced dissociation. Additionally, ion mobility spectrometry was carried out on selected species. Oligonucleotide duplexes of different sequences--bearing mismatch positions and abasic sites of complementary DNA 15-mers--were investigated to unravel general trends in their stability in the gas phase. Single-stranded LNA oligonucleotides were also investigated with respect to their gas phase behavior and fragmentation upon collision-induced dissociation. In contrast to the collision-induced dissociation of DNA, almost no base loss was observed for LNAs. Here, backbone cleavages were the dominant dissociation pathways. This finding was further underlined by the need for higher activation energies. Base losses from the LNA strand were also absent in fragmentation experiments of the investigated DNA-LNA hybrid duplexes. While DNA-DNA duplexes dissociated easily into single stranded fragments, the high stability of DNA-LNA hybrids resulted in predominant fragmentation of the DNA part rather than the LNA, while base losses were only observed from the DNA single strand of the hybrid. [ABSTRACT FROM AUTHOR]

Primary hypolactasia is the main cause of lactose intolerance in adults. It is strongly associated with the single genetic variant LCT-13910C>T, located upstream of the lactase encoding gene. Consequently, analysis of LCT-13910C>T has been recommended as a direct genetic test for the trait. The aim of our study was to develop a TaqMan probe based real-time PCR protocol for the detection of the LCT-13910C>T variant directly from whole blood, circumventing DNA isolation. The LCT-13910C>T variant was determined using the DirectBlood Genotyping PCR Kit (myPOLS Biotec, Konstanz, Germany) together with an in-house TaqMan primer-probe assay. Validity and specificity of the assay was evaluated using EDTA anti-coagulated whole blood samples and corresponding DNA samples. Results from real-time PCR were compared with results obtained by Sanger sequencing from 105 blinded whole blood samples. Validity and specificity of the assay using whole blood were comparable to those using purified genomic DNA as substrate in PCR. Genetic analysis of blood samples were in complete agreement with results obtained by Sanger sequencing. In conclusion, we present a reliable real-time PCR protocol for the detection of the LCT-13910C>T variant directly from whole blood further facilitating diagnosis of primary hypolactasia in symptomatic patients. [ABSTRACT FROM AUTHOR]

Alpha-l-Locked nucleic acid (α-l-LNA) is a stereoisomeric analogue of locked nucleic acid (LNA), which possesses excellent biophysical properties and also exhibits high target binding affinity to complementary oligonucleotide sequences and resistance to nuclease degradations. Therefore, α-l-LNA nucleotides could be utilised to develop stable antisense oligonucleotides (AO), which can be truncated without compromising the integrity and efficacy of the AO. In this study, we explored the potential of α-l-LNA nucleotides-modified antisense oligonucleotides to modulate splicing by inducing Dmd exon-23 skipping in mdx mouse myoblasts in vitro. For this purpose, we have synthesised and systematically evaluated the efficacy of α-l-LNA-modified 2′-O-methyl phosphorothioate (2′-OMePS) AOs of three different sizes including 20mer, 18mer and 16mer AOs in parallel to fully-modified 2′-OMePS control AOs. Our results demonstrated that the 18mer and 16mer truncated AO variants showed slightly better exon-skipping efficacy when compared with the fully-23 modified 2′-OMePS control AOs, in addition to showing low cytotoxicity. As there was no previous report on using α-l-LNA-modified AOs in splice modulation, we firmly believe that this initial study could be beneficial to further explore and expand the scope of α-l-LNA-modified AO therapeutic molecules.

Lampreys have a worldwide distribution, are functionally important to ecological communities and serve significant roles in many cultures. In Pacific coast drainages of North America, lamprey populations have suffered large declines. However, lamprey population status and trends within many areas of this region are unknown and such information is needed for advancing conservation goals. We developed two quantitative PCR-based, aquatic environmental DNA (eDNA) assays for detection of Pacific Lamprey (Entosphenus tridentatus) and Lampetra spp, using locked nucleic acids (LNAs) in the probe design. We used these assays to characterize the spatial distribution of lamprey in 18 watersheds of Puget Sound, Washington, by collecting water samples in spring and fall. Pacific Lamprey and Lampetra spp were each detected in 14 watersheds and co-occurred in 10 watersheds. Lamprey eDNA detection rates were much higher in spring compared to fall. Specifically, the Pacific Lamprey eDNA detection rate was 3.5 times higher in spring and the Lampetra spp eDNA detection rate was 1.5 times higher in spring even though larval lamprey are present in streams year-round. This significant finding highlights the importance of seasonality on eDNA detection. Higher stream discharge in the fall likely contributed to reduced eDNA detection rates, although seasonal life history events may have also contributed. These eDNA assays differentiate Pacific Lamprey and Lampetra spp across much of their range along the west coast of North America. Sequence analysis indicates the Pacific Lamprey assay also targets other Entosphenus spp and indicates the Lampetra spp assay may have limited or no capability of detecting Lampetra in some locations south of the Columbia River Basin. Nevertheless, these assays will serve as a valuable tool for resource managers and have direct application to lamprey conservation efforts, such as mapping species distributions, occupancy modeling, and monitoring translocations and reintroductions.

Background: Hybrid LC-MS assays for oligonucleotides rely on capture probes to develop assays with high sensitivity and specificity. Locked nucleic acid (LNA) probes are thermodynamically superior to existing capture probes, but are not currently used for hybrid LC-MS assays. Materials & methods: Using two lipid-conjugated double-stranded siRNA compounds as model analytes, hybrid LC-MS/MS assays using LNA probes were developed. Results : The workflows demonstrated the superiority of the LNA probes, optimized sample preparation conditions to maximize analyte recovery, evaluated the need for analyte-specific internal standards, and demonstrated that advanced mass spectrometric technology can increase assay sensitivity by up to 20-fold. Conclusion: The workflow can be used in future bioanalytical studies to develop effective hybrid LC-MS/MS methods for siRNA analytes.

The Ebola virus is a zoonotic pathogen that can cause severe hemorrhagic fever in humans, with up to 90% lethality. The deadly 2014 Ebola outbreak quickly made an unprecedented impact on human lives. While several vaccines and therapeutics are under development, current approaches contain several limitations, such as virus mutational escape, need for formulation or refrigeration, poor scalability, long lead-time, and high cost. To address these challenges, we developed locked nucleic acid (LNA)-modified antisense oligonucleotides (ASOs) to target critical Ebola viral proteins and the human intracellular host protein Niemann-Pick C1 (NPC1), required for viral entry into infected cells. We generated noninfectious viral luciferase reporter assays to identify LNA ASOs that inhibit translation of Ebola viral proteins in vitro and in human cells. We demonstrated specific inhibition of key Ebola genes VP24 and nucleoprotein, which inhibit a proper immune response and promote Ebola virus replication, respectively. We also identified LNA ASOs targeting human host factor NPC1 and demonstrated reduced infection by chimeric vesicular stomatitis virus harboring the Ebola glycoprotein, which directly binds to NPC1 for viral infection. These results support further in vivo testing of LNA ASOs in infectious Ebola virus disease animal models as potential therapeutic modalities for treatment of Ebola. [ABSTRACT FROM AUTHOR]

Lampreys have a worldwide distribution, are functionally important to ecological communities and serve significant roles in many cultures. In Pacific coast drainages of North America, lamprey populations have suffered large declines. However, lamprey population status and trends within many areas of this region are unknown and such information is needed for advancing conservation goals. We developed two quantitative PCR-based, aquatic environmental DNA (eDNA) assays for detection of Pacific Lamprey (Entosphenus tridentatus) and Lampetra spp, using locked nucleic acids (LNAs) in the probe design. We used these assays to characterize the spatial distribution of lamprey in 18 watersheds of Puget Sound, Washington, by collecting water samples in spring and fall. Pacific Lamprey and Lampetra spp were each detected in 14 watersheds and co-occurred in 10 watersheds. Lamprey eDNA detection rates were much higher in spring compared to fall. Specifically, the Pacific Lamprey eDNA detection rate was 3.5 times higher in spring and the Lampetra spp eDNA detection rate was 1.5 times higher in spring even though larval lamprey are present in streams year-round. This significant finding highlights the importance of seasonality on eDNA detection. Higher stream discharge in the fall likely contributed to reduced eDNA detection rates, although seasonal life history events may have also contributed. These eDNA assays differentiate Pacific Lamprey and Lampetra spp across much of their range along the west coast of North America. Sequence analysis indicates the Pacific Lamprey assay also targets other Entosphenus spp and indicates the Lampetra spp assay may have limited or no capability of detecting Lampetra in some locations south of the Columbia River Basin. Nevertheless, these assays will serve as a valuable tool for resource managers and have direct application to lamprey conservation efforts, such as mapping species distributions, occupancy modeling, and monitoring translocations and reintroductions. [ABSTRACT FROM AUTHOR]

Single nucleotide polymorphisms (SNPs) are single nucleotide variations which comprise the most wide spread source of genetic diversity in the genome. Currently, SNPs serve as markers for genetic predispositions, clinically evident disorders and diverse drug responses. Present SNP diagnostics are primarily based on enzymatic reactions in different formats including sequencing, polymerase-chain reaction (PCR) and microarrays. In these assays, the enzymes are applied to address the required sensitivity and specificity when detecting SNP. On the other hand, the development of enzyme-free, simple and robust SNP sensing methods is in a constant focus in research and industry as such assays allow rapid and reproducible SNP diagnostics without the need for expensive equipment and reagents. An ideal method for detection of SNP would entail mixing a DNA or RNA target with a probe to directly obtain a signal. Current assays are still not fulfilling these requirements, although remarkable progress has been achieved in recent years. In this review, current SNP sensing approaches are described with a main focus on recently introduced direct, enzyme-free and ultrasensitive SNP sensing by optical methods.

Oligonucleotides are key compounds widely used for research, diagnostics, and therapeutics. The rapid increase in oligonucleotide-based applications, together with the progress in nucleic acids research, has led to the design of nucleotide analogs that, when part of these oligomers, enhance their efficiency, bioavailability, or stability. One of the most useful nucleotide analogs is the first-generation bridged nucleic acids (BNA), also known as locked nucleic acids (LNA), which were used in combination with ribonucleotides, deoxyribonucleotides, or other analogs to construct oligomers with diverse applications. However, there is still room to improve their efficiency, bioavailability, stability, and, importantly, toxicity. A second-generation BNA, BNANC (2′-O,4′-aminoethylene bridged nucleic acid), has been recently made available. Oligomers containing these analogs not only showed less toxicity when compared to LNA-containing compounds but, in some cases, also exhibited higher specificity. Although there are still few applications where BNANC-containing compounds have been researched, the promising results warrant more effort in incorporating these analogs for other applications. Furthermore, newer BNA compounds will be introduced in the near future, offering great hope to oligonucleotide-based fields of research and applications.

Transcription of the HIV-1 provirus generates a viral pre-mRNA, which is alternatively spliced into more than 50 HIV-1 mRNAs encoding all viral proteins. Regulation of viral alternative splice site usage includes the presence of splicing regulatory elements (SREs) which can dramatically impact RNA expression and HIV-1 replication when mutated. Recently, we were able to show that two viral SREs, GI3-2 and ESEtat, are important players in the generation of viral vif, vpr and tat mRNAs. Furthermore, we demonstrated that masking these SREs by transfected locked nucleic acid (LNA) mixmers affect the viral splicing pattern and viral particle production. With regard to the development of future therapeutic LNA mixmer-based antiretroviral approaches, we delivered the GI3-2 and the ESEtat LNA mixmers “nakedly”, without the use of transfection reagents (gymnosis) into HIV-1 infected cells. Surprisingly, we observed that gymnotically-delivered LNA mixmers accumulated in the cytoplasm, and seemed to co-localize with GW bodies and induced degradation of mRNAs containing their LNA target sequence. The GI3-2 and the ESEtat LNA-mediated RNA degradation resulted in abrogation of viral replication in HIV-1 infected Jurkat and PM1 cells as well as in PBMCs.

Apolipoprotein C3 (Apo C3) LNA/DNA gapmer was evaluated under various stress and formulation conditions for the purpose of its development as a potential biotherapeutic for low density lipoprotein (LDL) lowering. Using ion-pairing (IP) reversed-phase (RP) liquid chromatography ultra-high resolution (UHR) tandem mass spectrometry (IP-RPLC-MS/MS), a combination of accurate mass measurements and collision-induced dissociation enabled in-depth characterization of Apo C3 LNA/DNA oligonucleotide, in particular the inherent impurities following synthesis and degradation products after exposure to stress conditions. In this study, oligonucleotide samples were stressed under different pH and UV exposure conditions. The primary impurities in Apo C3 LNA/DNA were losses of nucleotide moieties from both the 5′- and 3′-terminus leading to n-1, n-2, etc. species. Desulfurization and depurination were observed in Apo C3 LNA/DNA after a week under UV light stress conditions at low pH. Guanine oxidation and dimerization were the primary degradation products detected under UV light exposure for 1 week at high pH. The effect of antioxidants on the levels of these degradation products was evaluated under neutral pH conditions. In the presence of all antioxidants, levels of guanine oxidation and desulfurization under tested conditions were the same as those in the unstressed sample, except for sodium ascorbate. The thorough understanding of the Apo C3 LNA/DNA oligonucleotide structure, its impurities, and degradation products laid the foundation for the successful formulation development of this novel biotherapeutic modality. [ABSTRACT FROM AUTHOR]

In Neisseria gonorrhoeae , fluoroquinolone resistance is caused by mutations on various genes, especially on gyrA. Locked nucleic acid DNA sequence was added to the forward primer for gyrA and subjected to real-time quantitative polymerase chain reaction, enabling simultaneous detection of N. gonorrhoeae and mutations associated with antimicrobial resistance. • Quinolone-resistant gonorrhea incidence is increasing is difficult to diagnose. • Point mutations in the Neisseria gonorrhoeae gyrA gene cause quinolone resistance. • Locked nucleic acids bind to complementary nucleic acids. • Locked nucleic acids were added to primers and subjected to real-time PCR. • This simultaneously detected N. gonorrhoeae and antibiotic resistance mutations. [ABSTRACT FROM AUTHOR]

Velvet antlers are used medicinally in Asia and possess various therapeutic effects. Prices are set according to the country of origin, which is unidentifiable to the naked eye, and therefore counterfeiting is prevalent. Additionally, antlers of the Canadian elk, which can generate chronic wasting disease, are prevalently smuggled and distributed in the market. Thus, a method for identifying the country of origin of velvet antlers was developed, using polymorphisms in mitochondrial DNA, fluorescence melting curve analysis and analysis of locked nucleic acids (LNA). This combined method is capable of identifying five genotypes of velvet antlers in a single experiment using two probes. It also has advantages in multiplexing, simplicity and efficiency in genotyping, when compared to real-time PCR or microarrays. The developed method can be used to improve identification rates in the velvet antler market and, by extension, research based on polymorphisms in DNA sequences. [ABSTRACT FROM AUTHOR]

Fluorescence in situ hybridization (FISH) employing nucleic acid mimics as probes is becoming an emerging molecular tool in the microbiology area for the detection and visualization of microorganisms. However, the impact that locked nucleic acid (LNA) and 2′-O-methyl (2′-OMe) RNA modifications have on the probe that is targeting microorganisms is unknown. In this study, the melting and hybridization efficiency properties of 18 different probes in regards to their use in FISH for the detection of the 16S rRNA of Helicobacter pylori were compared. For the same sequence and target, probe length and the type of nucleic acid mimics used as mixmers in LNA-based probes strongly influence the efficiency of detection. LNA probes with 10 to 15 mers showed the highest efficiency. Additionally, the combination of 2′-OMe RNA with LNA allowed an increase on the fluorescence intensities of the probes. Overall, these results have significant implications for the design and applications of LNA probes for the detection of microorganisms. [ABSTRACT FROM AUTHOR]

Background When oligonucleotides hybridize to long target molecules, the process is slowed by the secondary structure in the targets. The phenomenon has been analyzed in several previous studies, but many details remain poorly understood. Methods I used a spectrofluorometric strategy, focusing on the formation/breaking of individual base pairs, to study the kinetics of association between a DNA hairpin and > 20 complementary oligonucleotides (‘antisenses’). Results Hybridization rates differed by over three orders of magnitude. Association was toehold-mediated, both for antisenses binding to the target's ends and for those designed to interact with the loop. Binding of these latter, besides being consistently slower, was affected to variable, non-uniform extents by the asymmetric loop structure. Divalent metal ions accelerated hybridization, more pronouncedly when nucleation occurred at the loop. Incorporation of locked nucleic acid (LNA) residues in the antisenses substantially improved the kinetics only when LNAs participated to the earliest hybridization steps. The effects of individual LNAs placed along the antisense indicated that the reaction transition state occurred after invading at least the first base pair of the stem. Conclusions The experimental approach helps dissect hybridization reactions involving structured nucleic acids. Toehold-dependent, nucleation–invasion models appear fully appropriate for describing such reactions. Estimating the stability of nucleation complexes formed at internal toeholds is the major hurdle for the quantitative prediction of hybridization rates. General significance While analyzing the mechanisms of a fundamental biochemical process (hybridization), this work also provides suggestions for the improvement of technologies that rely on such process. [ABSTRACT FROM AUTHOR]

The thermodynamics and kinetics of DNA hybridization, i.e. the process of self-assembly of one, two or more complementary nucleic acid strands, has been studied for many years. The appearance of the nearest-neighbor model led to several theoretical and experimental papers on DNA thermodynamics that provide reasonably accurate thermodynamic information on nucleic acid duplexes and allow estimation of the melting temperature. Because there are no thermodynamic models specifically developed to predict the hybridization temperature of a probe used in a fluorescencein situhybridization (FISH) procedure, the melting temperature is used as a reference, together with corrections for certain compounds that are used during FISH. However, the quantitative relation between melting and experimental FISH temperatures is poorly described. In this review, various models used to predict the melting temperature for rRNA targets, for DNA oligonucleotides and for nucleic acid mimics (chemically modified oligonucleotides), will be addressed in detail, together with a critical assessment of how this information should be used in FISH. [ABSTRACT FROM PUBLISHER]

Oligodeoxyribonucleotides modified with 5-[3-(1-pyrenecarboxamido)propynyl]-2′-deoxyuridine monomer X and proximal LNA monomers display higher affinity for complementary DNA, more pronounced increases in fluorescence emission upon DNA binding, and improved discrimination of SNPs at non-stringent conditions, relative to the corresponding LNA-free probes across a range of sequence contexts. The results reported herein suggest that the introduction of LNA monomers influences the position of nearby fluorophores via indirect conformational restriction, a characteristic that can be utilized to develop optimized fluorophore-labeled probes for SNP-discrimination studies. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are small regulatory RNAs, which mediate selective repression of gene expression. miRNAs play important roles in many natural and pathological processes. Numerous tools were developed for their detection and functional analysis. There are many excellent articles covering different areas of miRNA biology in detail. At the same time, I think there are many colleagues who face a miRNA-related research problem and would appreciate having an introductory general overview of tools for miRNA analysis, which would help them in considering available options. Accordingly, this review provides an elementary roadmap to navigate among available tools for miRNA analysis. The most common problems and errors observed in miRNA research are also discussed. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs), small RNA molecules that post-transcriptionally regulate mRNA expression, are crucial in diverse developmental and physiological programs and their misregulation can lead to disease. Chemically modified oligonucleotides have been developed to modulate miRNA activity for therapeutic intervention in disease settings, but their mechanism of action has not been fully elucidated. Here we show that the miRNA inhibitors (anti-miRs) physically associate with Argonaute proteins in the context of the cognate target miRNA in vitro and in vivo. The association is mediated by the seed region of the miRNA and is sensitive to the placement of chemical modifications. Furthermore, the targeted miRNAs are stable and continue to be associated with Argonaute. Our results suggest that anti-miRs specifically associate with Argonaute-bound miRNAs, preventing association with target mRNAs, which leads to subsequent stabilization and thus increased expression of the targeted mRNAs. [ABSTRACT FROM AUTHOR]

Abstract: A 20-mer phosphorothioate antisense oligodeoxyribonucleotide having locked nucleic acids (LNA–AON) was used to reduce elevated serum triglyceride levels in mice. We repeatedly administered LNA–AON, which targets murine apolipoprotein C-III mRNA, to high-fat-fed C57Bl/6J male mice for 2 weeks. The LNA–AON showed efficient dose-dependent reductions in hepatic apolipoprotein C-III mRNA and decreased serum apolipoprotein C-III protein concentrations, along with efficient dose-dependent reductions in serum triglyceride concentrations and attenuation of fat accumulation in the liver. Through precise lipoprotein profiling analysis of sera, we found that serum reductions in triglyceride and cholesterol levels were largely a result of decreased serum very low-density lipoprotein (VLDL)-triglycerides and -cholesterol. It is noteworthy that larger VLDL particles were more susceptible to removal from blood than smaller particles, resulting in a shift in particle size distribution to smaller diameters. Histopathologically, fatty changes were markedly reduced in antisense-treated mice, while moderate granular degeneration was frequently seen the highest dose of LNA–AON. The observed granular degeneration of hepatocytes may be associated with moderate elevation in the levels of serum transaminases. In conclusion, we developed an LNA-based selective inhibitor of apolipoprotein C-III. Although it remains necessary to eliminate its potential hepatotoxicity, the present LNA–AON will be helpful for further elucidating the molecular biology of apolipoprotein C-III. [Copyright &y& Elsevier]

MicroRNAs (miRNAs) are endogenous non-protein coding RNA molecules of approximately 21 nucleotides in length capable of modulating gene expression in animals and plants. The role of miRNA based gene regulation has been proved in several pathways including in plant growth, development and stress response. In this study miR171 and miR397a were tested for their expression pattern under different heat shock regimes in shoot and root tissues of Arabidopsis thaliana using Locked Nucleic Acid (LNA) mediated in situ hybridization. With an increase in temperature across 35 °C, 40 °C and 45 °C there was a corresponding increased up-regulation of miR171 in leaf tissues compared to ambient temperature. Similarly, an unambiguous elevated expression of miR171 within increase in duration of exposure at each temperature regime across 1 h, 2 h and 3 h was noticed in comparison to ambient control leaf tissue. On the other hand, miR397a, which expressed at ambient control conditions, got down-regulated both with increase in heat and exposure regime in leaf tissues. Both miRNAs expressed in control ambient root tissues. Maintaining the root zone temperature at ambient conditions, upon imposing heat shock regime to shoot system, miR171 recorded corresponding increased up-regulation as indicated by the intensity of in situ hybridization, while miR397a got down-regulated. Given the differential homogeneity in expression pattern of both miRNA in leaf and root tissues experiencing heat shock regimes, possibilities of movement of heat shock induced signals to root tissues seem to be obvious. [ABSTRACT FROM AUTHOR]

Background: Gilbert's syndrome is a common metabolic dysfunction characterized by elevated levels of unconjugated bilirubin in the bloodstream. This condition is usually caused by additional (TA) insertions in a promoter region of the uridine diphosphate glucuronosyltransferase 1A1 ( UGT1A1) gene, which instead of the sequence А(TА)6TАА contains А(TА)7TАА. While the condition itself is benign, it presents elevated risk for patients treated with irinotecan, a common chemotherapy drug. Methods: The technique is based on hybridization analysis of a pre-amplified segment of the UGT1A1 gene promoter performed on a microarray. Specific probes containing locked nucleic acids (LNA) were designed and immobilized on the microarray to provide accurate identification. Results: A microarray has been developed to identify both common and rare variants of UGT1A1(TA)n polymorphisms. In total, 108 individuals were genotyped. Out of these, 47 (43.5%) had homozygous wild-type genotypes (TA)6/(TA)6; 41(38%) were heterozygotes (TA)6/(TA)7; and 18 (16.7%) - homozygotes (TA)7/(TA)7. In two cases (1.8%), rare genotypes (TA)5/(TA)7and (TA)5/(TA)6were found. The results were in full agreement with the sequencing. In addition, synthetic fragments corresponding to all human allelic variants [(TA)5, (TA)6, (TA)7, (TA)8] were successfully tested. Conclusions: The developed microarray-based approach for identification of polymorphic variants of the UGT1A1 gene is a promising and reliable diagnostic tool that can be successfully implemented in clinical practice. [ABSTRACT FROM AUTHOR]