Efficient detection of secondary structure folded nucleic acids related to Alzheimer's disease based on junction probes

Single stranded DNA often forms stable secondary structures under physiological conditions. These DNA secondary structures play important physiological roles. However, the analysis of such secondary structure folded DNA is often complicated because of its high thermodynamic stability and slow hybridization kinetics. In this article, we demonstrate that Y-shaped junction probes could be used for rapid and highly efficient detection of secondary structure folded DNA. Our approach contained a molecular beacon (MB) probe and an assistant probe. In the absence of target, the MB probe failed to hybridize with the assistant probe. Whereas, the MB probe and the assistant probe could cooperatively unwind the secondary structure folded DNA target to form a ternary Y-shaped junction structure. In this condition, the MB probe was also opened, resulting in separating the fluorophores from the quenching moiety and emitting the fluorescence signal. This approach allowed for the highly sensitive detection of secondary structure folded DNA target, such as a tau specific DNA fragment related to Alzheimer's disease in this case. Additionally, this approach showed strong SNPs identifying capability. Furthermore, it was noteworthy that this newly proposed approach was capable of detecting secondary structure folded DNA target in cell lysate samples.     

The dibenzodioxocin lignin substructure is abundant in the inner part of the secondary wall in Norway spruce and silver birch xylem

A specific condensed lignin substructure, dibenzodioxocin, was immunolocalized in differentiating cell walls of Norway spruce (Picea abies (L.) H. Karsten) and silver birch (Betula pendula Roth) xylem. A fluorescent probe, Alexa 488 was used as a marker on the dibenzodioxocin-specific secondary antibody. For the detection of this lignin substructure, 25-m cross-sections of xylem were viewed with a confocal laser-scanning microscope with fluorescein isothiocyanate fluorescence filters. In mature cells, fluorescence was detected in the S3 layer of the secondary wall in both tree species, but it was more intense in Norway spruce than in silver birch. In silver birch most of the signal was detected in vessel walls and less in fiber cell walls. In very young tracheids of Norway spruce and vessels and fibers of silver birch, where secondary cell wall layers were not yet formed, the presence of the dibenzodioxocin structure could not be shown.Abbreviation CLSM confocal laser-scanning fluorescence microscopy     

In situ hybridization of Prochlorococcus and Synechococcus (marine cyanobacteria) spp. with RRNA-targeted peptide nucleic acid probes

A simple method for whole-cell hybridization using fluorescently labeled rRNA-targeted peptide nucleic acid (PNA) probes was developed for use in marine cyanobacterial picoplankton. In contrast to established protocols, this method is capable of detecting rRNA in Prochlorococcus, the most abundant unicellular marine cyanobacterium. Because the method avoids the use of alcohol fixation, the chlorophyll content of Prochlorococcus cells is preserved, facilitating the identification of these cells in natural samples. PNA probe-conferred fluorescence was measured flow cytometrically and was always significantly higher than that of the negative control probe, with positive/negative ratio varying between 4 and 10, depending on strain and culture growth conditions. Prochlorococcus cells from open ocean samples were detectable with this method. RNase treatment reduced probe-conferred fluorescence to background levels, demonstrating that this signal was in fact related to the presence of rRNA. In another marine cyanobacterium, Synechococcus, in which both PNA and oligonucleotide probes can be used in whole-cell hybridizations, the magnitude of fluorescence from the former was fivefold higher than that from the latter, although the positive/negative ratio was comparable for both probes. In Synechococcus cells growing at a range of growth rates (and thus having different rRNA concentrations per cell), the PNA- and oligonucleotide-derived signals were highly correlated (r = 0.99). The chemical nature of PNA, the sensitivity of PNA-RNA binding to single-base-pair mismatches, and the preservation of cellular integrity by this method suggest that it may be useful for phylogenetic probing of whole cells in the natural environment.     

Desulfotomaculum genus- and subgenus-specific 16S rRNA hybridization probes for environmental studies

Based on comparative analysis of 16S rRNA sequences and the recently established phylogeny of the genus Desulfotomaculum , a set of phylogenetically nested hybridization probes was developed and characterized. A genus-specific probe targets all known Desulfotomaculum species (with the exception of Desulfotomaculum acetoxidans ), and five specific probes target subclusters within the Desulfotomaculum genus. The dissociation temperature of each probe was determined experimentally. Probe specificities were verified through hybridizations with pure culture rRNA isolated from a wide variety of target and non-target organisms and through an evaluation of probe 'nesting' using samples obtained from four different environments. Fixation and hybridization conditions for fluorescence in situ hybridizations were also optimized. The probes were used in quantitative membrane hybridizations to determine the abundance of Desulfotomaculum species in thermophilic anaerobic digesters, in soil, in human faeces and in pig colon samples. Desulfotomaculum rRNA accounted for 0.3–2.1% of the total rRNA in the digesters, 2.6–6.6% in soil, 1.5–3.3% in human faeces and 2.5–6.2% in pig colon samples.     

Salt dependent changes in structure and dynamics of circular single stranded DNA of filamentous phages of Escherichia coli

We have analyzed the static and dynamic behaviour of the circular single stranded DNA of the filamentous Escherichia coli phages F1 and M13mp8 in solution as a function of salt concentration using static and dynamic light scattering and sedimentation analysis in the analytical ultracentrifuge. We show by static light scattering that native and denatured single stranded DNA behave like a randomly coiled macromolecule at all salt concentrations used. The size of the native single stranded DNA is governed by the formation of secondary structures. While the radius of gyration decreases with increasing salt concentration the translational diffusion of the center-of-mass of native single stranded DNA and the sedimentation coefficient increase with increasing salt concentration in a biphasic manner. Below 100 mM monovalent cation concentration there is a strong dependence of the hydrodynamic parameters upon salt which is reduced approx. 3-fold at higher salt concentrations. We attribute the compaction of single stranded DNA by salt to electrostatic shielding and, in case of native single stranded DNA, secondary structure formation. Internal motions of the native single stranded DNA are observable at all salt concentrations and can be interpreted with a model of segmental diffusion of the elements of the polymer chain. The observed segmental diffusion coefficient of the native single stranded polynucleotide increases with increasing salt under the conditions investigated.     

Developing aptamer probes for acute myelogenous leukemia detection and surface protein biomarker discovery

Background

The majority of patients with acute myelogenous leukemia (AML) still die of their disease. In order to improve survival rates in AML patients, new strategies are necessary to discover biomarkers for the detection and targeted therapy of AML. One of the advantages of the aptamer-based technology is the unique cell-based selection process, which allows us to efficiently select for cell-specific aptamers without knowing which target molecules are present on the cell surface.

Methods

The NB4 AML cell line was used as the target cell population for selecting single stranded DNA aptamers. After determining the affinity of selected aptamers to leukocytes, the aptamers were used to phenotype human bone marrow leukocytes and AML cells in clinical specimens. Then a biotin-labelled aptamer was used to enrich and identify its target surface protein.

Results

Three new aptamers were characterized from the selected aptamer pools (JH6, JH19, and K19). All of them can selectively recognize myeloid cells with Kd in the low nanomole range (2.77 to 12.37 nM). The target of the biotin-labelled K19 aptamer probe was identified as Siglec-5, a surface membrane protein in low abundance whose expression can serve as a biomarker of granulocytic maturation and be used to phenotype AML. More importantly, Siglec-5 expression can be used to detect low concentrations of AML cells in human bone marrow specimens, and functions as a potential target for leukemic therapy.

Conclusions

We have demonstrated a pipeline approach for developing single stranded DNA aptamer probes, phenotyping AML cells in clinical specimens, and then identifying the aptamer-recognized target protein. The developed aptamer probes and identified Siglec-5 protein may potentially be used for leukemic cell detection and therapy in our future clinical practice.     

A simple method for monitoring protein–DNA interactions

A simple, efficient and cheap method is reported for monitoring interactions between single stranded desoxyribonucleic acids and proteins, using fluorescence spectroscopy and complexes of 5′-dye–DNA conjugates with bovine serum albumin as probes. In the presence of a single stranded DNA-binding protein the complexes with bovine serum albumin are disrupted, which results in a reduction of fluorescence intensity.     

A reduction-triggered fluorescence probe for sensing nucleic acids

We have developed a reduction-triggered fluorescence probe with a new fluorogenic compound derivatized from Rhodamine for sensing oligonucleotides. The chemistry to activate the compound involves the reaction between the azide group of rhodamine derivatives and the reducing reagents, with the fluorescence signal appearing after reduction of the azide group. The signal/background ratio of this fluorogenic compound reached 2100-fold enhancement in fluorescence intensity. Dithio-1,4-threitol or triphenylphosphine as reducing reagents were successfully utilized for this chemistry to be introduced into the DNA probe. The genetic detection requires that two strands of DNA bind onto target oligonucleotides, one probe carrying a reducible fluorogenic compound while the other carries the reducing reagents. The reaction proceeds automatically without any enzymes or reagents under biological conditions to produce a fluorescence signal within 10-20 min in the presence of target DNA or RNA. In addition, the probe was very stable under biological conditions, even such extreme conditions as pH 5 solution, pH 10 solution, or high temperature (90 degrees C) with no undesirable background signal. The probes were successfully applied to the detection of oligonucleotides at the single nucleotide level in solution and endogenous RNA in bacterial cells.     

Detection of single-copy functional genes in prokaryotic cells by two-pass TSA-FISH with polynucleotide probes

In situ detection of functional genes with single-cell resolution is currently of interest to microbiologists. Here, we developed a two-pass tyramide signal amplification (TSA)-fluorescence in situ hybridization (FISH) protocol with PCR-derived polynucleotide probes for the detection of single-copy genes in prokaryotic cells. The mcrA gene and the apsA gene in methanogens and sulfate-reducing bacteria, respectively, were targeted. The protocol showed bright fluorescence with a good signal-to-noise ratio and achieved a high efficiency of detection (> 98%). The discrimination threshold was approximately 82-89% sequence identity. Microorganisms possessing the mcrA or apsA gene in anaerobic sludge samples were successfully detected by two-pass TSA-FISH with polynucleotide probes. The developed protocol is useful for identifying single microbial cells based on functional gene sequences.     

Real-time multiplex PCR assays

The ability to multiplex PCR by probe color and melting temperature (T(m)) greatly expands the power of real-time analysis. Simple hybridization probes with only a single fluorescent dye can be used for quantification and allele typing. Different probes are labeled with dyes that have unique emission spectra. Spectral data are collected with discrete optics or dispersed onto an array for detection. Spectral overlap between dyes is corrected by using pure dye spectra to deconvolute the experimental data by matrix algebra. Since fluorescence is temperature dependent and depends on the dye, spectral overlap and color compensation constants are also temperature dependent. Single-labeled probes are easier to synthesize and purify than more complex probes with two or more dyes. In addition, the fluorescence of single-labeled probes is reversible and depends only on hybridization of the probe to the target, allowing study of the melting characteristics of the probe. Although melting curves can be obtained during PCR, data are usually acquired at near-equilibrium rates of 0.05-0.2 degrees C/s after PCR is complete. Using rapid-cycle PCR, amplification requires about 20 min followed by a 10-min melting curve, greatly reducing result turnaround time. In addition to dye color, melting temperature can be used for a second dimension of multiplexing. Multiplexing by color and T(m) creates a "virtual" two-dimensional multiplexing array without the need for an immobilized matrix of probes. Instead of physical separation along the X and Y axes, amplification products are identified by different fluorescence spectra and melting characteristics.     

Obstacles to flow cytometric analysis of rumen microbial samples

Several methods were tested that would improve the fluorescence signal from hybridized rumen bacterial cells. Disruption of cell envelopes by lysozyme, EDTA, proteinase K and/or SDS caused only a minor increase in fluorescence signal. Use of helper unlabeled oligonucleotide probes was successful only with the Puni[H672] probe which, however, when used with specific PBBl4-labeled probe, gave fluorescence signal drop. No substantial rise in fluorescence signal was also observed with cells subjected to growth-without-cell-division treatment. Further improvements are needed to make the fluorescent in situ hybridization (FISH)-flow cytometry combination applicable to rumen bacteria.     

Quenched autoligation probes allow discrimination of live bacterial species by single nucleotide differences in rRNA

Quenched autoligation (QUAL) probes are a class of self-reacting nucleic acid probes that give strong fluorescence signal in the presence of fully complementary RNAs and selectivity against single nucleotide differences in solution. Here, we describe experiments designed to test whether QUAL probes can discriminate between bacterial species by the detection of small differences in their 16S rRNA sequences. Probes were introduced into live cells using small amounts of detergent, thus eliminating the need for fixation, and fluorescence signal was monitored both by microscopy and by flow cytometry without any washing steps. The effects of probe length, modified backbone, probe concentration and growth state of the bacteria were investigated. The data demonstrate specific fluorescence discrimination between three closely related bacteria, Escherichia coli, Salmonella enterica and Pseudomonas putida, based on single nucleotide differences in their 16S rRNA. Discrimination was possible with cells in mid-log phase or in lag phase. These results suggest that QUAL probes may be useful for rapid identification of microorganisms in laboratory and clinical settings.     

Unlabeled helper oligonucleotides increase the in situ accessibility to 16S rRNA of fluorescently labeled oligonucleotide probes

Target site inaccessibility represents a significant problem for fluorescence in situ hybridization (FISH) of 16S rRNA with oligonucleotide probes. Here, unlabeled oligonucleotides (helpers) that bind adjacent to the probe target site were evaluated for their potential to increase weak probe hybridization signals in Escherichia coli DSM 30083(T). The use of helpers enhanced the fluorescence signal of all six probes examined at least fourfold. In one case, the signal of probe Eco474 was increased 25-fold with the use of a single helper probe, H440-2. In another case, four unlabeled helpers raised the FISH signal of a formerly weak probe, Eco585, to the level of the brightest monolabeled oligonucleotide probes available for E. coli. The temperature of dissociation and the mismatch discrimination of probes were not significantly influenced by the addition of helpers. Therefore, using helpers should not cause labeling of additional nontarget organisms at a defined stringency of hybridization. However, the helper action is based on sequence-specific binding, and there is thus a potential for narrowing the target group which must be considered when designing helpers. We conclude that helpers can open inaccessible rRNA regions for FISH with oligonucleotide probes and will thereby further improve the applicability of this technique for in situ identification of microorganisms.     

Application of rRNA-based probes for observing marine nanoplanktonic protists.

The use of small-subunit rRNA-based oligonucleotides as probes for detecting marine nanoplanktonic protists was examined with a ciliate (an Uronema sp.), a flagellate (a Cafeteria sp.), and mixed assemblages of protists from enrichment cultures and natural seawater samples. Flow cytometry and epifluorescence microscopy analyses demonstrated that hybridizations employing fluorescein-labeled, eukaryote-specific probes intensely stained logarithmically growing protists, whereas these same protist strains in late stationary growth were barely detectable. The fluorescence intensity due to probe binding was significantly enhanced by the use of probes end labeled with biotin, which were detected by fluorescein-labeled avidin. The degree of signal amplification ranged from two- to fivefold for cultured protists in both logarithmic and stationary growth phases. Mixed assemblages of heterotrophic protists from enrichment cultures were also intensely labeled by rRNA-targeted oligonucleotide probes by the biotin-avidin detection system. Protists in late stationary growth phase and natural assemblages of protists that were otherwise undetectable when hybridized with fluorescein-labeled probes were easily visualized by this approach. In the latter samples, hybridization with multiple, biotin-labeled probes was necessary for detection of naturally occurring marine protists by epifluorescence microscopy. The signal amplification obtained with the biotin-avidin system should increase the utility of rRNA-targeted probes for identifying protists and facilitate characterization of the population structure and distribution of protists in aquatic environments.     

The relative rotation of the ends of Bacillus subtilis during growth

Observation of long single filaments of Bacillus subtilis 168 in depression slide cultures demonstrated that one end rotated relative to the other during growth. This was observed with suspended filaments, filaments attached to glass surfaces and single stranded filaments folded back on themselves growing as a double stranded helix. This extends Mendelson's 1976 conclusion to cases with no alternative interpretation to the hypothesis that as each cell grows, the structure of the peptidoglycan changes to rotate one end relative to the other.     

Plant cell culture monitoring using an in situ multiwavelength fluorescence probe

A multiwavelength fluorescence probe is proposed for in situ monitoring of Eschscholtzia californica and Catharanthus roseus plant cell cultures. The potential of the probe as a tool for real-time estimation of biomass and production in secondary metabolites has been studied. The probe excitation range is 270-550 nm and the emission range is 310-590 nm, with a step of 20 nm for both excitation and emission filters. Many endogenous fluorophores such as NAD(P)H, riboflavins (riboflavin and derivatives such as FMN, FAD), tryptamine and tryptophan, and fluorescent secondary metabolites were analyzed simultaneously. NAD(P)H fluorescence signal (350/450 nm) showed to be an adequate signal for estimating cells activity. Riboflavins fluorescence signal (450/530 nm) followed C. roseus cell concentration both for the growth phase and after elicitation with jasmonic acid. Fluorescence from the alkaloids interfered with NAD(P)H signal during the production phase. For C. roseus, tryptophan, tryptamine, ajmalicine and serpentine were monitored by the probe. For E. californica, fluorescence from alkaloids overlapped with riboflavins preventing from using the probe to follow cell growth but global alkaloids production could be followed using the probe.