LABELING DURING CLEAVAGE (LDC), A NEW LABELING APPROACH FOR RNA

A new and efficient strategy for labeling of RNA sequences prior to their hybridization on high density DNA chip has been developed. Our approach which combines the fragmentation and the labeling is based on the reactivity of the 3′-phosphate of cleaved RNA fragments with a fluorescent molecule bearing aromatic bromomethyl function.     

Labeling during cleavage (LDC), a new labeling approach for RNA

A new and efficient strategy for labeling of RNA sequences prior to their hybridization on high density DNA chip has been developed. Our approach which combines the fragmentation and the labeling is based on the reactivity of the 3'-phosphate of cleaved RNA fragments with a fluorescent molecule bearing aromatic bromomethyl function.     

Labeling during cleavage of nucleic acids for their detection on DNA chips

A new and efficient strategy for labeling of nucleic acids prior to their hybridization on high density DNA chip has been developed. Our approach which combines the fragmentation and the labeling is based on the reactivity of the terminal phosphates of cleaved DNA and RNA fragments with a reporter molecule bearing aryldiazomethane group.     

Labeling During Cleavage of Nucleic Acids for Their Detection on DNA Chips

Abstract

A new and efficient strategy for labeling of nucleic acids prior to their hybridization on high density DNA chip has been developed. Our approach which combines the fragmentation and the labeling is based on the reactivity of the terminal phosphates of cleaved DNA and RNA fragments with a reporter molecule bearing aryldiazomethane group.     

Nonradioactive labeling of probe with digoxigenin by polymerase chain reaction

Probes nonradioactively labeled with the steroid hapten digoxigenin have several intriguing properties, including a high sensitivity equivalent to that of radioactive probes, speed in detection, low hazard potential in handling, and possibility of long-term storage. The use of polymerase chain reaction for labeling probe has been demonstrated to offer various advantages including efficient labeling of fragments as small as 100 bp, direct labeling of genomic DNA, and labeling with subnanogram amounts of input DNA. We therefore investigated whether this technique could be adapted for labeling with a relatively large molecule such as digoxigenin. In this report, we show that the polymerase chain reaction is a very efficient technique for synthesis of digoxigenin-labeled DNA and we present an extremely simple procedure for purification of the non-isotopically labeled fragments.     

Chemical methods of DNA and RNA fluorescent labeling.

Several procedures have been described for fluorescent labeling of DNA and RNA. They are based on the introduction of aldehyde groups by partial depurination of DNA or oxidation of the 3'-terminal ribonucleoside in RNA by sodium periodate. Fluorescent labels with an attached hydrazine group are efficiently coupled with the aldehyde groups and the hydrazone bonds are stabilized by reduction with sodium cyanoborohydride. Alternatively, DNA can be quantitatively split at the depurinated sites with ethylenediamine. The aldimine bond between the aldehyde group in depurinated DNA or oxidized RNA and ethylenediamine is stabilized by reduction with sodium cyanoborohydride and the primary amine group introduced at these sites is used for attachment of isothiocyanate or succinimide derivatives of fluorescent dyes. The fluorescent DNA labeling can be carried out either in solution or on a reverse phase column. These procedures provide simple, inexpensive methods of multiple DNA labeling and of introducing one fluorescent dye molecule per RNA, as well as quantitative DNA fragmentation and incorporation of one label per fragment. These methods of fluorophore attachment were shown to be efficient for use in the hybridization of labeled RNA, DNA and DNA fragments with oligonucleotide microchips.     

Efficient fluorescence labeling of a large RNA through oligonucleotide hybridization

We present an efficient method of introducing fluorophore labels at selected locations in a large RNA. The method is based on specific and highly efficient hybridization between a fluorophore-containing DNA oligonucleotide and a modular hairpin loop replacing a functionally unimportant hairpin loop in the RNA. We demonstrate its feasibility using a 255-nucleotide RNA derived from the catalytic domain of RNase P from Bacillus subtilis. Hybridization of the DNA oligonucleotide to the modular hairpin loop minimally perturbs the structure and function of this RNA. This labeling scheme should be applicable in studies of RNA conformational dynamics by ensemble and single molecule fluorescence methods.     

Four-color single-molecule fluorescence with noncovalent dye labeling to monitor dynamic multimolecular complexes

To enable studies of conformational changes within multimolecular complexes, we present a simultaneous, four-color single molecule fluorescence methodology implemented with total internal reflection illumination and camera-based, wide-field detection. We further demonstrate labeling histidine-tagged proteins noncovalently with Tris-nitrilotriacetic acid (Tris-NTA)-conjugated dyes to achieve single molecule detection. We combine these methods to colocalize the mismatch repair protein MutSα on DNA while monitoring MutSα-induced DNA bending using F?rster resonance energy transfer (FRET) and to monitor assembly of membrane-tethered SNARE protein complexes.     

Use of halogenated precursors for simultaneous DNA and RNA detection by means of immunoelectron and immunofluorescence microscopy.

We have developed a novel approach for in situ labeling and detection of nucleic acids in cultured cells. It is based on in vivo incorporation of chlorouridine (ClU) or iododeoxyuridine (IdU) into Chinese hamster ovary cells with the aim of labeling RNA and DNA, respectively. The halogenated nucleotides are immunolabeled on ultrathin sections with anti-bromodeoxyuridine (BrdU) monoclonal antibodies that specifically react with either IdU or ClU. Furthermore, we combined ClU and IdU incubation to label simultaneously RNA and DNA in the same cell. Both were visualized by means of anti-BrdU antibodies exhibiting strong affinity for one of the two halogenated epitopes. Confocal imaging of interphase nuclei and electron microscopic analysis showed evidence of a partial colocalization of newly synthesized DNA and RNA inside the cell nucleus. RNase and DNase digestion of ultrathin sections after formaldehyde fixation and acrylic resin embedding confirmed the specificity of incorporation. Consequently, this method allows us to differentially label DNA and RNA on the same section. Using short pulses with the precursors, we could show that newly synthesized DNA and RNA both preferentially occur within the perichromatin region at the border of condensed chromatin domains.     

Selective labeling and detection of specific RNAs in an RNA mixture

We report here a unique approach to selectively label and detect specific RNA in an RNA mixture (without separation or purification) using DNA polymerase, dNTP labels, and a short synthetic DNA template complementary to the 3(')-terminus of the RNA. The detection sensitivity is high, at attomole level (10-18 mole). The selective principle was demonstrated by individually labeling and detecting RNAs in a RNA mixture when different templates were provided. By taking advantage of the template-directed selectivity, poly(A) tail-containing mRNA in total RNA was detected and labeled at the 3(')-terminal on a poly(T) template. Nonradioactive labels, such as fluorophore and antigen labels, may also be used; this method can be applied in methodology for direct detection and quantification of viral RNAs.     

Nicking enzyme-based internal labeling of DNA at multiple loci

The labeling of biomolecules has become standard practice in molecular biosciences. Modifications are used for detection, sorting and isolation of small molecules, complexes and entire cells. We have recently reported a method for introducing internal chemical and structural modifications into kbp-sized DNA target substrates that are frequently used in single-molecule experiments. It makes use of nicking enzymes that create single-stranded DNA gaps, which can be subsequently filled with labeled oligonucleotides. Here we provide a detailed protocol and further expand this method. We show that modifications can be introduced at distant loci within one molecule in a simple one-pot reaction. In addition, we achieve labeling on both strands at a specific locus, as demonstrated by F?rster resonance energy transfer (FRET) experiments. The protocol requires an initial cloning of the target substrate (3-5 d), whereas the labeling itself takes 4-6 h. More elaborate purification and verification of label incorporation requires 2 h for each method.     

A DNAzyme based label-free detection system for miniaturized assays

Sensitive detection assays are a prerequisite for the analysis of small amounts of samples derived from biological material. There is a great demand for highly sensitive and robust detection techniques to analyze biomolecules. The combination of catalytic active DNA (DNAzyme) with a peroxidase activity with rolling circle amplification (RCA) is a promising alternative to common detection systems. The rolling circle amplification leads to a product with tandemly linked copies of DNAzymes. The continuous signal generation of the amplified DNAzymes results in an increased sensitivity. The combination of two amplification reactions, namely RCA and DNAzymes, results in increased signal intensity by a factor of 10(6). With this approach the labeling of samples can be avoided. The advantage of the introduced assay is the usage of nucleic acids as biosensors for the detection of biomolecules. Coupling of the analyte molecule to the detection molecules allows the direct detection of the analyte molecule. The described label-free hotpot assay has a broad potential field of applications. The hotpot assay can be adapted to detect and analyze RNA, DNA and proteins down to femtomolar concentrations in a miniaturized platform with a total reaction solution of 50 nl. The applicability of the assay for diagnostics and research will be shown with a focus on high throughput systems using a nano-well platform.     

Multi-nanopore force spectroscopy for DNA analysis

The need for low-cost DNA sequence detection in clinical applications is driving development of new technologies. We demonstrate a method for detection of mutations in a DNA sequence purely by electronic means, and without need for fluorescent labeling. Our method uses an array of nanopores to perform synchronized single-molecule force spectroscopy measurements over many molecules in parallel, yielding detailed information on the kinetics of hundreds of molecule dissociations in a single measurement.     

Multiplexed, particle-based detection of DNA using flow cytometry with 3DNA dendrimers for signal amplification.

BACKGROUND: Complex mixtures of DNA may be found in environmental and medical samples. There is a need for techniques that can measure low concentrations of target DNAs. For a multiplexed, flow cytometric assay, we show that the signal-to-noise ratio for fluorescence detection may be increased with the use of 3DNA dendrimers. A single fluorescent DNA molecule per bead could be detected with conventional flow cytometry instrumentation. METHODS: The analyte consisted of single-stranded (ss) DNA amplicons that were hybridized to capture probes on the surface of fluorescent polystyrene microspheres (beads) and initially labeled with streptavidin-R-phycoerythrin (single-step labeling). These beads have a low reporter fluorescence background and high efficiency of DNA hybridization. The DNA/SA-RPE complex was then labeled with 3DNA dendrimers and SA-RPE. The bead complexes were detected with a Luminex 100 flow cytometer. Bead standards were developed to convert the intensity to the number of SA-RPE labels per bead and the number of dendrimers per bead. RESULTS: The dendrimer assay resulted in 10-fold fluorescence amplification compared with single-step SA-RPE labeling. Based on concentration curves of pure target ss-amplicons, the signal-to-noise ratio of the dendrimer assay was greater by a factor of 8.5 over single-step SA-RPE labeling. The dendrimer assay was tested on 16S ribosomal DNA amplified from filter retentates of contaminated groundwater. Multiplexed detection of a single dendrimer-labeled DNA molecule per bead was demonstrated. CONCLUSIONS: Multiplexed detection of DNA hybridization on a single molecule level per bead was achieved with conventional flow cytometry instrumentation. This assay is useful for detecting target DNAs at low concentrations.     

<Emphasis Type="Italic">In situ</Emphasis>detection of non-polyadenylated RNA molecules using Turtle Probes and target primed rolling circle PRINS

Background  

In situ detection is traditionally performed with long labeled probes often followed by a signal amplification step to enhance the labeling. Whilst short probes have several advantages over long probes (e.g. higher resolution and specificity) they carry fewer labels per molecule and therefore require higher amplification for detection. Furthermore, short probes relying only on hybridization for specificity can result in non-specific signals appearing anywhere the probe attaches to the target specimen. One way to obtain high amplification whilst minimizing the risk of false positivity is to use small circular probes (e.g. Padlock Probes) in combination with target primed rolling circle DNA synthesis. This has previously been used for DNA detection in situ, but not until now for RNA targets.     

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5’-ATCGAT-3’ sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.     

In situ hybridization at the electron microscope level: hybrid detection by autoradiography and colloidal gold

In situ hybridization has become a standard method for localizing DNA or RNA sequences in cytological preparations. We developed two methods to extend this technique to the transmission electron microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope in situ hybridization. Radioactively labeled complementary RNA (cRNA) is hybridized to metaphase chromosomes deposited on electron microscope grids and fixed in 70 percent ethanol vapor; hybridixation site are detected by autoradiography. Specific and intense labeling of chromosomal centromeric regions is observed even after relatively short exposure times. Inerphase nuclei present in some of the metaphase chromosome preparations also show defined paatterms of satellite DNA labeling which suggests that satellite-containing regions are associate with each other during interphase. The sensitivity of this method is estimated to at least as good as that at the light microscope level while the resolution is improved at least threefold. The second method, which circumvents the use of autoradiogrphic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction is improved at least threefold. The second method, which circumvents the use of autoradiographic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction with an antibody against biotin and secondary antibody adsorbed to the surface of over centromeric heterochromatin and along the associated peripheral fibers. Labeling is on average ten times that of background binding. This method is rapid and possesses the potential to allow precise ultrastructual localization of DNA sequences in chromosomes and chromatin.