A Sensitive Alternative for MicroRNA In Situ Hybridizations Using Probes of 2��-O-Methyl RNA + LNA

The use of short, high-affinity probes consisting of a combination of DNA and locked nucleic acid (LNA) has enabled the specific detection of microRNAs (miRNAs) by in situ hybridization (ISH). However, detection of low–copy number miRNAs is still not always possible. Here the authors show that probes consisting of 2′-O-methyl RNAs (2OMe) and LNA at every third base (2:1 ratio), under optimized hybridization conditions, excluding yeast RNA from the hybridization buffer, can provide superior performance in detection of miRNA targets in terms of sensitivity and signal-to-noise ratio compared to DNA + LNA probes. Furthermore, they show that hybridizations can be performed in buffers of 4M urea instead of 50% formamide, thereby yielding an equally specific but nontoxic assay. The use of 2OMe + LNA–based probes and the optimized ISH assay enable simple and fast detection of low–copy number miRNA targets, such as miR-130a in mouse brain.     

LNA-modified oligonucleotides are highly efficient as FISH probes

Fluorescence in situ hybridization (FISH) is a highly useful technique with a wide range of applications including the delineation of complex karyotypes, prenatal diagnosis of aneuploidies, screening for diagnostic or prognostic markers in cancer cells, gene mapping and gene expression studies. However, it is still a fairly time-consuming method with limitations in both sensitivity and resolution. Locked Nucleic Acids (LNAs) constitute a novel class of RNA analogs that have an exceptionally high affinity towards complementary DNA and RNA. Substitution of DNA oligonucleotide probes with LNA has shown to significantly increase their thermal duplex stability as well as to improve the discrimination between perfectly matched and mismatched target nucleic acids. To exploit the improved hybridization properties of LNA oligonucleotides in FISH, we have designed several LNA substituted oligonucleotide probes specific to different human-specific repetitive elements, such as the classical satellite-2, telomere and alpha-satellite repeats. In the present study we show that LNA modified oligonucleotides are excellent probes in FISH, combining high binding affinity with short hybridization time.     

Cleavage and protection of locked nucleic acid-modified DNA by restriction endonucleases

Locked nucleic acid (LNA) is one of the most prominent nucleic acid analogues reported so far. We herein for the first time report cleavage by restriction endonuclease of LNA-modified DNA oligonucleotides. The experiments revealed that RsaI is an efficient enzyme capable of recognizing and cleaving LNA-modified DNA oligonucleotides. Furthermore, introduction of LNA nucleotides protects against cleavage by the restriction endonucleases PvuII, PstI, SacI, KpnI and EcoRI.     

Chemical Synthesis of LNA-mCTP and its application for MicroRNA detection

Locked nucleic acids (LNA) are being applied in hybridization studies, but current locked nucleotides cannot be transcribed into RNA probes. Here, the authors report the use of a new synthetic locked nucleotide, locMeCytidine-5'-triphosphate (LNA-mCTP), for hybridization study. This synthetic LNA-mCTP can be transcribed into a short ( approximately 30-nt) RNA probe. Dot blot hybridization on nylon membrane suggested that the short (33)P-LNA RNA probes had strong binding affinity to target oligonucleotides and its detection sensitivity was approximately approximately 1000 miRNAs in a 20- to 30-mum (diameter) dot area. On tissue sections, the differential expression pattern of mir-124 within different tissue regions revealed by short (33)P- LNA RNA probes correlated well to that analyzed by real-time RT-PCR. In addition, the specific cellular distribution of vasoactive intestinal polypeptide mRNAs in the mouse brain was the same using a 30-nt (33)P-LNA RNA probe and a 1.5-kb (33)P-RNA probe. These results suggested the high hybridization specificity of the small LNA-RNA probes to target small RNAs. Finally, the authors applied (33)P-LNA probes to detect miRNA let-7C expression in human cancer tissues. Let-7C was clearly present in lung, prostate, and colon cancers but undetectable in ovary and thyroid cancer samples. These results suggested that this miRNA detection method provides an alternative tool to study the cellular distribution of miRNAs in tissues.     

LNA-enhanced detection of single nucleotide polymorphisms in the apolipoprotein E

Genotyping of single nucleotide polymorphisms (SNPs) in large populations presents a great challenge, especially if the SNPs are embedded in GC-rich regions, such as the codon 112 SNP in the human apolipoprotein E (apoE). In the present study, we have used immobilized locked nucleic acid (LNA) capture probes combined with LNA-enhancer oligonucleotides to obtain efficient and specific interrogation of SNPs in the apoE codons 112 and 158, respectively. The results demonstrate the usefulness of LNA oligonucleotide capture probes combined with LNA enhancers in mismatch discrimination. The assay was applied to a panel of patient samples with simultaneous genotyping of the patients by DNA sequencing. The apoE genotyping assays for the codons 112 and 158 SNPs resulted in unambiguous results for all patient samples, concurring with those obtained by DNA sequencing.     

“Locked onto the target”: increasing the efficiency of antagomirzymes using locked nucleic acid modifications

This study highlights the effect of incorporation of locked nucleic acid (LNA) on improving the functional efficacy of DNAzymes against microRNAs (antagomirzymes). DNAzymes were designed against two different sites of miR-27a, which were encompassed both within the precursor and mature form of miRNA. The cleavage and functional activities of these DNAzymes have been compared to those of LNA-modified counterparts, containing LNA modification in each of the substrate binding arms. Preliminary examination based on in vitro cleavage demonstrated LNAzyme to be much more effective in the ensuing cleavage of target miRNA under both single- and multiple-turnover conditions. Evaluation of kinetic parameters indicated almost 5-fold higher cleavage efficiency, kobs, for LNAzymes than for DNAzymes, leading to more efficient cleavage of the substrate. We attribute this enhancement in cleavage efficiency to the LNA-mediated improvement in the hybridization of the antagomirzyme·target complex. Functional validation of the relative activities was accomplished through the luciferase reporter assay and quantitative real-time polymerase chain reaction (qRT-PCR). Both the unmodified and LNA-modified antagomirzymes were very active in ensuing efficient miRNA knockdown; however, compared to the DNAzymes, the LNAzymes were almost 25% more active. A direct quantitative estimate of miRNA cleavage, conducted using qRT-PCR, further substantiated the data by indicating that LNAzyme effectively downregulated the levels of mature miRNA (up to 50%) versus the corresponding DNAzymes. Our data thus provide formative evidence of the successful employment of LNA-based antagomirzymes against miRNA.     

Dramatically improved RNA in situ hybridization signals using LNA-modified probes

In situ detection of RNA by hybridization with complementary probes is a powerful technique. Probe design is a critical parameter in successful target detection. We have evaluated the efficiency of fluorescent DNA oligonucleotides modified to contain locked nucleic acid (LNA) residues. This increases the thermal stability of hybrids formed with RNA. The LNA-based probes detect specific RNAs in fixed yeast cells with an efficiency far better than conventional DNA oligonucleotide probes of the same sequence. Using this probe design, we were also able to detect poly(A)(+) RNA accumulation within the nucleus/ nucleolus of wild-type cells. LNA-based probes should be readily applicable to a diverse array of cells and tissue samples.     

LNA probes substantially improve detection of bacterial endosymbionts in whole mount of insects by fluorescent in-situ hybridization

ABSTRACT: BACKGROUND: Detection of unculturable bacteria and their localization in the host, by fluorescent in-situ hybridization (FISH), is a powerful technique in the study of host-bacteria interaction. FISH probes are designed to target the 16 s rRNA region of the bacteria to be detected. LNA probes have recently been used in FISH studies and proven to be more efficient. To date no report has employed LNA probes for FISH detection of bacterial endosymbiont in the whole mount tissues. Further, though speculated, bacteriocytes have not been reported from males of Bemisia tabaci. RESULTS: In this study, we compared the efficiency in detecting bacteria by fluorescent DNA oligonucleotides versus modified probes containing Locked Nucleic Acid (LNA) substitution in their structure. We used the insect Bemisia tabaci as the experimental material since it carried simultaneous infection by two bacteria: one a primary endosymbiont, Portiera (and present in more numbers) while the other a secondary endosymbiont Arsenophonus (and present in less numbers). Thus a variation in the abundance of bacteria was expected. While detecting both the bacteria, we found a significant increase in the signal whenever LNA probes were used. However, the difference was more pronounced in detecting the secondary endosymbiont, wherein DNA probes gave weak signals when compared to LNA probes. Also, signal to noise ratio for LNA probes was higher than DNA probes. We found that LNA considerably improved sensitivity of FISH, as compared to the commonly used DNA oligonucleotide probe. CONCLUSION: By employing LNA probes we could detect endosymbiotic bacteria in males, which have never been reported previously. We were able to detect bacteriocytes containing Portiera and Arsenophonus in the males of B. tabaci. Thus, employing LNA probes at optimized conditions will help to significantly improve detection of bacteria at the lowest concentration and may give a comprehensible depiction about their specific distribution within samples.     

Locked nucleic acid-based in situ detection of microRNAs in mouse tissue sections

Here we describe a method for sensitive and specific histological detection of microRNAs (miRNAs) by in situ hybridization. The protocol focuses on the use of locked nucleic acids (LNAs), which are bi-cyclic RNA analogs that allow a significant increase in the hybridization temperature and thereby an enhanced stringency for short probes as required for miRNA detection. The protocol is optimized for cryosections in order to study the spatial and temporal expression of miRNAs with high sensitivity and resolution. We detail how to construct probes, set up and conduct an LNA in situ hybridization experiment. In addition, we discuss alternative colorimetric strategies that can be used to effectively detect and visualize miRNAs including double staining with other markers. Setting up and conducting the in situ experiment is estimated to take approximately 1 week, assuming that all the component parts are readily available.     

LNA flow–FISH: A flow cytometry–fluorescence in situ hybridization method to detect messenger RNA using locked nucleic acid probes

We present a novel method using flow cytometry–fluorescence in situ hybridization (flow–FISH) to detect specific messenger RNA (mRNA) in suspended cells using locked nucleic acid (LNA)-modified oligonucleotide probes. β-Actin mRNA was targeted in whole A549 epithelial cells by hybridization with a biotinylated, LNA-modified probe. The LNA bound to β-actin was then stained using phycoerythrin-conjugated streptavidin and detected by flow cytometry. Shifts in fluorescence signal intensity between the β-actin LNA probe and a biotinylated, nonspecific control LNA were used to determine optimal conditions for this type of flow–FISH. Multiple conditions for permeabilization and hybridization were tested, and it was found that conditions using 3 μg/ml of proteinase K for permeabilization and 90 min hybridization at 60 °C with buffer containing 50% formamide allow cells containing the LNA-bound mRNA to be detected and differentiated from the control LNA with high confidence (< 14% overlap between curves). This combined method, called LNA flow–FISH, can be used for detection and quantification of other RNA species as well as for telomerase measurement and detection.     

Real-time genotyping with oligonucleotide probes containing locked nucleic acids

Oligonucleotide probes containing locked nucleic acid (LNA) hybridize to complementary single-stranded target DNA sequences with an increased affinity compared to oligonucleotide DNA probes. As a consequence of the incorporation of LNA residues into the oligonucleotide sequence, the melting temperature of the oligonucleotide increases considerably, thus allowing the successful use of shorter LNA probes as allele-specific tools in genotyping assays. In this article, we report the use of probes containing LNA residues for the development of qualitative fluorescent multiplex assays for the detection of single nucleotide polymorphisms (SNPs) in real-time polymerase chain reaction using the 5'-nuclease detection assay. We developed two applications that show the improved specificity of LNA probes in assays for allelic discrimination. The first application is a four-color 5'-nuclease assay for the detection of SNPs for two of the most common genetic factors involved in thrombotic risk, factor V Leiden and prothrombin G20210A. The second application is a two-color assay for the specific detection of the A-to-T tranversion in codon 6 of the beta-globin gene, responsible for sickle cell anemia. Both real-time genotyping assays were evaluated by comparing the performance of our method to that of a reference method and in both cases, we found a 100% concordance. This approach will be useful for research and molecular diagnostic laboratories in situations in which the specificity provided by oligonucleotide DNA probes is insufficient to discriminate between two DNA sequences that differ by only one nucleotide.     

Liquid-based hybridization assay with real-time detection in miniaturized array platforms

An assay for the fluorescent detection of short oligonucleotide probe hybridization in miniaturized high-density array platforms is presented. It combines hybridization in solution with real-time fluorescent detection, which involves measurement of fluorescence increase by means of an induced fluorescence resonance energy transfer. The feasibility of this approach using DNA or RNA as a target, and short DNA- as well as LNA (locked nucleic acid)-modified oligonucleotides as probes is shown. The presented approach could potentially contribute to a significant increase in the throughput of large-scale genomic applications, such as oligofingerprinting and genotyping, and also reduce material consumption.     

miRNA Expression Analyses in Prostate Cancer Clinical Tissues

A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, diagnosis, prognosis and treatment. In recent years, microRNAs (miRNAs) have emerged as promising alternate biomarkers for prostate cancer diagnosis and prognosis. However, the development of miRNAs as effective biomarkers for prostate cancer heavily relies on their accurate detection in clinical tissues. miRNA analyses in prostate cancer clinical specimens is often challenging owing to tumor heterogeneity, sampling errors, stromal contamination etc. The goal of this article is to describe a simplified workflow for miRNA analyses in archived FFPE or fresh frozen prostate cancer clinical specimens using a combination of quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH). Within this workflow, we optimize the existing methodologies for miRNA extraction from FFPE and frozen prostate tissues and expression analyses by Taqman-probe based miRNA RT-PCR. In addition, we describe an optimized method for ISH analyses formiRNA detection in prostate tissues using locked nucleic acid (LNA)- based probes. Our optimized miRNA ISH protocol can be applied to prostate cancer tissue slides or prostate cancer tissue microarrays (TMA).     

Using Amino-Labeled Nucleotide Probes for Simultaneous Single Molecule RNA-DNA FISH

Using amino-labeled oligonucleotide probes, we established a simple, robust and low-noise method for simultaneous detection of RNA and DNA by fluorescence in situ hybridization, a highly useful tool to study the large pool of long non-coding RNAs being identified in the current research. With probes either chemically or biologically synthesized, we demonstrate that the method can be applied to study a wide range of RNA and DNA targets at the single-cell and single-molecule level in cellular contexts.