Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Rapid genotyping using pyrene-perylene locked nucleic acid complexes.

ABSTRACT: We have developed an assay for single strand DNA and RNA detection which is based on novel pyrene-perylene FRET pairs attached to short LNA/DNA probes. The assay is based on ratiometric emission upon binding of target DNA/RNA by three combinations of fluorescent LNA/DNA reporter strands. Specific geometry of the pyrene fluorophore attached to the 2'-amino group of 2'-amino-LNA in position 4 allows for the first time to efficiently utilize dipole-dipole orientation parameter for sensing of single-nucleotide polymorphisms (SNPs) in nucleic acid targets by FRET. Using novel probes, SNP detection is achieved with advantages of large Stokes shift (115 nm), high fluorescence quantum yields and low limit of target detection values (< 5 nM). Rapid and accurate genotyping of highly polymorphic HIV Pol cDNA and RNA fragments performed herein proves the possibility for broad application of the novel pyrene-perylene FRET pairs, e.g., in imaging and clinical diagnostics.

SUBMITTER: Kumar TS

PROVIDER: S-EPMC3771999 | biostudies-literature | 2013 Apr-Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Rapid genotyping using pyrene-perylene locked nucleic acid complexes.

Kumar T Santhosh TS Myznikova Anna A Samokhina Evgeniya E Astakhova Irina Kira IK

Artificial DNA, PNA & XNA 20130401 2

We have developed an assay for single strand DNA and RNA detection which is based on novel pyrene-perylene FRET pairs attached to short LNA/DNA probes. The assay is based on ratiometric emission upon binding of target DNA/RNA by three combinations of fluorescent LNA/DNA reporter strands. Specific geometry of the pyrene fluorophore attached to the 2'-amino group of 2'-amino-LNA in position 4 allows for the first time to efficiently utilize dipole-dipole orientation parameter for sensing of single ...[more]

PMID: 24044052

Similar Datasets

Rapid <i>IDH1</i>-R132 genotyping panel utilizing locked nucleic acid loop-mediated isothermal amplification.

Project description:While the detection of single-nucleotide variants (SNVs) is important for evaluating human health and disease, most genotyping methods require a nucleic acid extraction step and lengthy analytical times. Here, we present a protocol which utilizes the integration of locked nucleic acids (LNAs) into self-annealing loop primers for the allelic discrimination of five isocitrate dehydrogenase 1 R132 (IDH1-R132) variants using loop-mediated isothermal amplification (LAMP). This genotyping panel was initially evaluated using purified synthetic DNA to show proof of specific SNV discrimination. Additional evaluation using glioma tumor lysates with known IDH1-R132 mutational status demonstrated specificity in approximately 35 min without the need for a nucleic acid extraction purification step. This LNA-LAMP-based genotyping assay can detect single base differences in purified nucleic acids or tissue homogenates, including instances where the variant of interest is present in an excess of background wild-type DNA. The pH-based colorimetric indicator of LNA-LAMP facilitates convenient visual interpretation of reactions, and we demonstrate successful translation to an end-point format using absorbance ratio, allowing for an alternative and objective approach for differentiating between positive and negative reactions. Importantly, the LNA-LAMP genotyping panel is highly reproducible, with no false-positive or false-negative results observed.

| S-EPMC10984729 | biostudies-literature

Novel locked nucleic acid (LNA)-based probe for the rapid identification of Chlamydia suis using real-time PCR.

Project description:BackgroundAs the importance of chlamydial infections in pigs has become more obvious, a rapid and sensitive method to study the prevalence of Chlamydia suis in pig herds is required. Such a method should permit routine diagnostic tests for herds with clinical and subclinical infections, without the need for Chlamydia culture.ResultsThe main objective of this study was to develop a specific and rapid method for detecting C. suis in swine herds. A real-time PCR assay using a single locked nucleic acid (LNA)-containing probe specific for C. suis was developed based on the previously described 28S rDNA fragment used to identify Chlamydiales. Use of LNA nucleotides enabled the single probe to target a short, specific fragment of the 23S rRNA. The probe showed high specificity for C. suis and did not show any cross-reactivity with other Chlamydia or Chlamydophila species nor with swine DNA. All of the 86 tested field isolates, earlier identified as C. suis, were confirmed as positive using the newly developed assay.ConclusionsUsing single LNA-based C. suis-specific probe allowed rapid and simple identification of this pathogen without requiring sequencing analysis and culturing. The proposed method may be used to study the prevalence of C. suis infection in pig herds and as a routine diagnostic test for herds with clinical and subclinical infection.

| S-EPMC4177436 | biostudies-literature

Probing 3D Collective Cancer Invasion Using Double-Stranded Locked Nucleic Acid Biosensors.

Project description:Cancer is a leading cause of death worldwide and metastases are responsible for over 90% of human cancer deaths. There is an urgent need to develop novel therapeutics for suppressing cancer invasion, the initial step of metastasis. Nevertheless, the regulation of cancer invasion is poorly understood due to a paucity of tools for monitoring the invasion process in 3D microenvironments. Here, we report a double-stranded locked nucleic acid (dsLNA) biosensor for investigating 3D collective cancer invasion. By incorporating multiphoton microscopy and the dsLNA biosensor, we perform dynamic single cell gene expression analysis while simultaneously characterizing the biomechanical interaction between the invading sprouts and the extracellular matrix. Gene profiling of invasive leader cells and detached cells suggest distinctive signaling mechanisms involved in collective and individual invasion in the 3D microenvironment. Our results underscore the involvement of Notch signaling in 3D collective cancer invasion, which warrants further investigation toward antimetastasis therapy in the future.

| S-EPMC5488859 | biostudies-literature

Mapping photothermally induced gene expression in living cells and tissues by nanorod-locked nucleic acid complexes.

Project description: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.

| S-EPMC4004321 | biostudies-literature

A novel approach for microRNA in situ hybridization using locked nucleic acid probes.

Project description:Identification of target tissue microRNAs (miR) using in situ hybridization (ISH), with digoxigenin-labeled locked nucleic acid (LNA) probes, is influenced by preanalytic parameters. To determine the best retrieval method for common microRNAs, a multiblock composed of paraffin-embedded tonsil, cervix, placenta, and hyperplastic prostate tissue were included. Tissue were fixed in 10% formalin in a range of 5-144 hours (h). Cut sections (5 μm) from the multiblock were subjected to combinations of pretreatment procedures: variable periods of proteinase K (PK) digestion or Heat-induced microRNA Retrieval (HmiRR) using target retrieval solution (TRS) pH 6.1 or 9, with or without enzymatic treatment (pepsin). Results for the overall categories: TRS pH 9 versus PK; p = 2.9e-23, TRS pH 9 versus TRS pH 6.1; p = 1.1e-14, TRS pH 6.1 versus PK; p = 2.9e-03. A long fixation time, resulted in the best microRNA preservation and staining intensity (long vs. short: p = 3.5e-47, long vs. moderate: p = 1.6e-44, moderate vs. short: p = 4.3e-16), was enhanced using HmiRR TRS pH 9 with or without pepsin providing high sensitivity and specificity. These observations conflict with other ISH techniques (e.g., messenger ribonucleic acid), which typically require shorter fixation periods, and therefore, further studies are warranted.

| S-EPMC7904755 | biostudies-literature

Accurate prediction of protein-nucleic acid complexes using RoseTTAFoldNA.

Project description:Protein-RNA and protein-DNA complexes play critical roles in biology. Despite considerable recent advances in protein structure prediction, the prediction of the structures of protein-nucleic acid complexes without homology to known complexes is a largely unsolved problem. Here we extend the RoseTTAFold machine learning protein-structure-prediction approach to additionally predict nucleic acid and protein-nucleic acid complexes. We develop a single trained network, RoseTTAFoldNA, that rapidly produces three-dimensional structure models with confidence estimates for protein-DNA and protein-RNA complexes. Here we show that confident predictions have considerably higher accuracy than current state-of-the-art methods. RoseTTAFoldNA should be broadly useful for modeling the structure of naturally occurring protein-nucleic acid complexes, and for designing sequence-specific RNA and DNA-binding proteins.

| S-EPMC10776382 | biostudies-literature

Down-modulation of cancer targets using locked nucleic acid (LNA)-based antisense oligonucleotides without transfection.

Project description:Usually, small interfering RNAs and most antisense molecules need mechanical or chemical delivery methods to down-modulate the targeted mRNA. However, these delivery approaches complicate the interpretations of biological consequences. We show that locked nucleic acid (LNA)-based antisense oligonucleotides (LNA-ONs) readily down-modulate genes of interest in multiple cell lines without any delivery means. The down-modulation of genes was quick, robust, long-lasting and specific followed by potent down-modulation of protein. The efficiency of the effect varied among the 30 tumor cell lines investigated. The most robust effects were found in those cells where nuclear localization of the LNA-ON was clearly observed. Importantly, without using any delivery agent, we demonstrated that HER3 mRNA and protein could be efficiently down-modulated in cells and a tumor xenograft model. These data provide a simple and efficient approach to identify potential drug targets and animal models. Further elucidation of the mechanism of cellular uptake and trafficking of LNA-ONs may enhance not only the therapeutic values of this platform but also antisense molecules in general.

| S-EPMC3154478 | biostudies-literature

Locked Nucleic Acid Probe-Based Real-Time PCR Assay for the Rapid Detection of Rifampin-Resistant Mycobacterium tuberculosis.

Project description:Drug-resistant Mycobacterium tuberculosis can be rapidly diagnosed through nucleic acid amplification techniques by analyzing the variations in the associated gene sequences. In the present study, a locked nucleic acid (LNA) probe-based real-time PCR assay was developed to identify the mutations in the rpoB gene associated with rifampin (RFP) resistance in M. tuberculosis. Six LNA probes with the discrimination capability of one-base mismatch were designed to monitor the 23 most frequent rpoB mutations. The target mutations were identified using the probes in a "probe dropout" manner (quantification cycle = 0); thus, the proposed technique exhibited superiority in mutation detection. The LNA probe-based real-time PCR assay was developed in a two-tube format with three LNA probes and one internal amplification control probe in each tube. The assay showed excellent specificity to M. tuberculosis with or without RFP resistance by evaluating 12 strains of common non-tuberculosis mycobacteria. The limit of detection of M. tuberculosis was 10 genomic equivalents (GE)/reaction by further introducing a nested PCR method. In a blind validation of 154 clinical mycobacterium isolates, 142/142 (100%) were correctly detected through the assay. Of these isolates, 88/88 (100%) were determined as RFP susceptible and 52/54 (96.3%) were characterized as RFP resistant. Two unrecognized RFP-resistant strains were sequenced and were found to contain mutations outside the range of the 23 mutation targets. In conclusion, this study established a sensitive, accurate, and low-cost LNA probe-based assay suitable for a four-multiplexing real-time PCR instrument. The proposed method can be used to diagnose RFP-resistant tuberculosis in clinical laboratories.

| S-EPMC4657947 | biostudies-literature

Application of Locked Nucleic Acid Oligonucleotides for siRNA Preclinical Bioanalytics.

Project description:Despite the exquisite potential of siRNA as a therapeutic, the mechanism(s) responsible for the robust indirect exposure-response relationships have not been fully elucidated. To understand the siRNA properties linked to potent activity, requires the disposition of siRNA to be characterized. A technical challenge in the characterization is the detection and quantitation of siRNA from biological samples. Described herein, a Locked Nucleic Acid (LNA) Hybridization-Ligation ECL ELISA was designed for ultra-sensitive quantification of both sense and antisense strands of siRNA independent of structural modifica-tions. This assay was applied to measure siRNA in serum and tissue homogenate in preclinical species. We observed rapid clearance of siRNA from the systemic circulation which contrasted the prolonged accumulation within the tissue. The assay was also able to distinguish and quantify free siRNA from RNA-induced silencing complex (RISC) and Argonaute 2 (Ago2) associated with therapeutic siRNA. We utilized an orthogonal method, LC-MS, to investigate 3' exonuclease activity toward the antisense strand metabolism. Taken together, we have demonstrated that the LNA Hybridization-Ligation ECL ELISA is arobust analytical method with direct application to measuring the exposure of siRNA therapeutics seamlessly across biological matrices.

| S-EPMC6401054 | biostudies-literature

The crystal structure of an 'All Locked' nucleic acid duplex.

Project description:'Locked nucleic acids' (LNAs) are known to introduce enhanced bio- and thermostability into natural nucleic acids rendering them powerful tools for diagnostic and therapeutic applications. We present the 1.9 Å X-ray structure of an 'all LNA' duplex containing exclusively modified β-D-2'-O-4'C-methylene ribofuranose nucleotides. The helix illustrates a new type of nucleic acid geometry that contributes to the understanding of the enhanced thermostability of LNA duplexes. A notable decrease of several local and overall helical parameters like twist, roll and propeller twist influence the structure of the LNA helix and result in a widening of the major groove, a decrease in helical winding and an enlarged helical pitch. A detailed structural comparison to the previously solved RNA crystal structure with the corresponding base pair sequence underlines the differences in conformation. The surrounding water network of the RNA and the LNA helix shows a similar hydration pattern.

| S-EPMC2965234 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data