Fluorescent Oligonucleotide Probes Research Articles

Abstract 4479: Polymorphisms in the nucleotide excision repair genes, ERCC1 and ERCC2/XPD and carcinogen DNA adducts in human lung

Abstract Objectives Excision repair cross-complementing group 1 (ERCC1) and excision repair cross-complementing group 2/xeroderma pigmentosum group D (ERCC2/XPD) in the nucleotide excision repair (NER) pathways that removes DNA damage caused by tobacco smoking carcinogen such as polycyclic aromatic hydrocarbons (PAHs). However, no studies have examined their role in DNA adducts in target human lung tissue, a dosimeter for lung cancer risk. Therefore, we investigated whether the polymorphisms in SNPs in NER pathways associated with the DNA adduct formation in human lung tissue. We further analyzed these relationship divided by adenocarcinoma and squamous cell carcinoma, main type of non-small cell lung cancer (NSCLC). Methods The study population consisted of 135 lung cancer patients from the Massachusetts General Hospital (MGH). Genotyping was completed for SNPs in ERCC1 [C8092A (rs3212986) and C118T (rs11615)] and ERCC2/XPD [Asp312Asn (rs1799793) and Lys751Gln (rs1052559)] using PCR-RFLP method and the PCR with fluorescent allele-specific oligonucleotide probes (TaqMan method). DNA adduct levels were measured as relative adduct levels per 1010 nucleotides by 32P-postlabeling in the lung. Multiple regression models were used to estimate adjusted percent change in DNA adduct levels associated with genotypes of the NER genes ERCC1 and ERCC2/XPD. Results After adjusting for potential confounders, DNA adduct levels in lung increased by 73.5% [95% confidence interval (CI), –22.6 to 288.9] for the ERCC2/XPD rs1799793AA genotype and by 43.7% (95% CI, –30.0 to 194.6) for ERCC2/XPD rs1052559GG genotype compared with their corresponding wild type homozygous genotypes in overall lung cancer patients, but it did not reach statistical significance. We found that DNA adducts levels in lung increased by 166.6% (95% CI, –5.0 to 648.3, P = 0.07) for ERCC2/XPD rs1799793AA genotype in patients with squamous cell carcinoma and the trend was borderline significant (P for trend = 0.06). Conclusions Polymorphisms of DNA repair genes, ERCC2/XPD Asp312Asn, may affect increased level of DNA adducts in the lung. Further large scale studies are needed to confirm our findings. Supported by NIH grants CA074386, CA092824, and CA90578. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4479. doi:1538-7445.AM2012-4479

Detection of Helicobacter pylori in raw bovine milk by fluorescence in situ hybridization (FISH)

The transmission pathways of Helicobacter pylori in humans have not been fully elucidated. Research in the last decade has proposed that foodborne transmission, among others, may be a plausible route of human infection. Owing to the organism's fastidious growth characteristics and its ability to convert to viable, yet unculturable states upon exposure to stress conditions, the detection of H. pylori in foods via culture-dependent methods has been proven to be laborious, difficult and in most cases unsuccessful. Hence, nucleic acid-based methods have been proposed as alternative methods but, to date, only PCR-based methods have been reported in the literature. In the current study, fluorescence in situ hybridization (FISH) was used for the detection of H. pylori in raw, bulk-tank bovine milk. After repeated milk centrifugation and washing steps, the bacterial flora of raw milk was subjected to fixation and permeabilization and H. pylori detection was conducted by FISH after hybridization with an H. pylori-specific 16S rRNA-directed fluorescent oligonucleotide probe. Using this protocol, H. pylori was detected in four out of the twenty (20%) raw milk samples examined. The data presented in this manuscript indicate that FISH can serve as an alternative molecular method for screening raw bovine milk for the presence of H. pylori.

Cy5-Conjugated Hybridization-Sensitive Fluorescent Oligonucleotides for Ratiometric Analysis of Nuclear Poly(A)+ RNA

Subnuclear poly(A)(+) RNA localization in living mammalian cells was visualized by ratiometric analysis using hybridization-sensitive fluorescent oligonucleotide probes. Probes were oligonucleotides, which contained a Cy5 fluorescent dye at the strand end and a thiazole orange double-labeled nucleotide inside strand. A ratiometric analysis using poly(A)-targeting probes revealed a distribution of the probe itself as red fluorescence and localization of the target RNA sequence in cell nuclei as green fluorescence. The fluorescence of the subnuclear poly(A)(+) RNA hybridized with the poly(A)-targeting probes was observed as puncta in interchromatin areas.

Highly Sensitive and Selective Bifunctional Oligonucleotide Probe for Homogeneous Parallel Fluorescence Detection of Protein and Nucleotide Sequence

The existing isothermal polymerization-based signal amplification assays are usually accomplished via two strategies: rolling circle amplification (RCA) and circular strand-displacement polymerization. In essence, the two techniques are based on cyclical nucleic acid strand-displacement polymerization (CNDP), limiting the application of isothermal polymerization in medical diagnosis and bioanalysis. In the present study, circular common target molecule (non-nucleic acid strand)-displacement polymerization (CCDP) is developed to amplify the fluorescence signal for biomolecule assays, extending isothermal polymerization to an aptameric system without any medium. Via combining an aptamer with a common hairpin DNA probe, we designed a self-blocked fluorescent bifunctional oligonucleotide probe (signaling probe) for the homogeneous parallel detection of two disease markers, PDGF-BB and the p53 gene. On the basis of CNDP and CCDP signal amplification, highly sensitive (e.g., detecting PDGF down to the concentration level of 1.8 × 10(-10) M) and selective detection (no interference even in the presence of a significantly higher concentration (7-200 times) of nontarget proteins) was accomplished, and the linear response range was considerably widened. Furthermore, the bifunctional signaling probe exhibits impressive simplicity, convenience, and short detection time. Herein, the design of the signaling probe was described, factors influencing fluorescence signal were investigated, analytical properties were characterized in detail, and the assay application in a complex medium was validated. The proposed biosensing scheme as a proof-of-concept is expected to promote the application of oligonucleotide probes in basic research and medical diagnosis.

LNA for Optimization of Fluorescent Oligonucleotide Probes: Improved Spectral Properties and Target Binding

Mixmer LNA/DNA fluorescent probes containing the 1-(phenylethynyl)pyrene fluorophore attached to 2'-arabino-uridine were synthesized and studied. The conjugates displayed significantly higher hybridization affinity to target DNA, increased fluorescence quantum yields of single-stranded oligonucleotides and their duplexes, and improved ability to form an interstrand excimer compared to analogous non-LNA probes.

Fluorescence Imaging of Influenza Virus H1N1 mRNA in Living Infected Cells using Single Chromophore FIT-PNA

Replication of the influenza virus involves, amongst other critical steps, the synthesis of viral mRNA which is used for ribosomal synthesis of viral proteins. Significant efforts have been devoted to the development of methods that allow the imaging of specific mRNA sequences in living cells. In contrast to other approaches, fluorescent oligonucleotide probes enable the analysis of non-modified wild-type targets. For imaging the probes must recognize the target with high specificity and deliver measurable signals that provide high signal-to-background ratios. We have introduced single labelled peptide nucleic acid (PNA) probes, so-called FIT-PNA probes, which contain a single thiazole orange intercalator that serves as artificial fluorescent nucleobase. These probes respond to changes of the local structure in the vicinity of the dye rather than to the more global changes of conformation that are required for fluorescence signalling by the dual labelled molecular beacons. FIT-probes are unique because a single fluorophore provides for both high sensitivity and high target specificity at non-stringent hybridization conditions where both matched and mismatched probe-target complexes coexist. We show that FIT-probes are ideally suited for applications in live cell RNA imaging. We chose mRNA coding for neuraminidase of influenza virus A/PR/8 as a target. FIT-PNA probes are useful to detect the expression of viral mRNA in single living infected cells with high specificity. In particular, FIT-PNA sensitivity was superior to that of a molecular beacon. Our study suggests that FIT-PNAs are attractive tools to study gene regulation of processes in living cells. The approach can be readily applied to other viruses as well.

A novel single-labeled fluorescent oligonucleotide probe for silver(i) ion detection based on the inherent quenching ability of deoxyguanosines

A novel single-labeled fluorescent oligonucleotide (OND) probe for the detection of nanomolar silver(I) ion in aqueous solution is developed based on the inherent quenching ability of deoxyguanosines. The formation of a hairpin structure of the OND-Ag(+) complex brings deoxyguanosines close to a dye, leading to a decreased fluorescence intensity of the dye owning to photoinduced electron transfer from the dye to deoxyguanosines.

ECHO probes: a concept of fluorescence control for practical nucleic acid sensing

An excitonic interaction caused by the H-aggregation of fluorescent dyes is a new type of useful photophysical process for fluorescence-controlled nucleic acid sensing. This critical review points out the recent advances in exciton-controlled hybridization-sensitive fluorescent oligonucleotide (ECHO) probes, which have a fluorescence-labeled nucleotide in which two molecules of thiazole orange or its derivatives are linked covalently. ECHO probes show absorption shift and emission switching depending on hybridization with the target nucleic acid. The hybridization-sensitive fluorescence emission of ECHO probes and the further modification of probes have made possible a variety of practical applications, such as multicolor RNA imaging in living cells and facile detection of gene polymorphism (144 references).

A Novel Single-Labeled Fluorescent Oligonucleotide Probe for Mercury(II) Ion Detection: Using the Inherent Quenching of Deoxyguanosines

A novel single-labeled fluorescent oligonucleotide (OND) probe for the detection of nanomolar mercury(II) ion in aqueous solution is developed based on the inherent quenching of deoxyguanosines. The formation of hairpin structure of OND-Hg(2+) complex brings deoxyguanosines close to dye, resulting in decreased dye fluorescence due to photoinduced electron transfer from dye to deoxyguanosines.

ECHO-LNA Conjugates: Hybridization-Sensitive Fluorescence and Its Application to Fluorescent Detection of Various RNA Strands

Hybridization-sensitive fluorescent DNA probes containing the nucleotide units of locked nucleic acid (LNA) have been developed. Exciton-controlled hybridization-sensitive fluorescent oligonucleotide (ECHO) probes that incorporated LNA nucleotides achieved high thermostability of the hybrid with target RNA strands. The appropriately designed ECHO-LNA chimeric probes exhibited an effective on-off switching property of fluorescence depending on hybridization with RNA and facilitated fluorescent detection of the TAR RNA strand forming a hairpin structure and distinction of one base difference in PLAC4 RNA sequence.

ON-OFF switching of transcriptional activity of large DNA through a conformational transition in cooperation with phospholipid membrane.

We report that structural transitions of DNA cause the ON−OFF switching of transcriptional activity in cooperation with phospholipid membrane in a reconstituted artificial cell. It has been shown that long DNA of more than 20−30 kilo base-pairs exhibits a discrete conformational transition between a coiled state and highly folded states in aqueous solution, depending on the presence of various condensing agents such as polyamine. Recently, we reported a conformational transition of long DNA through interplay with phospholipid membrane, from a folded state in aqueous phase to an extended coil state on a membrane surface, in a cell-sized water-in-oil microdroplet covered by phosphatidylethanolamine monolayer (Kato, A.; Shindo, E.; Sakaue, T.; Tsuji, A.; Yoshikawa, K. Biophys. J.2009, 132, 1678−1686). In this study, to elucidate the effects of these conformational changes on the biologically important function of DNA, transcription, we investigated the transcriptional activity of DNA in a microdroplet. Transcriptional activity was evaluated at individual DNA molecule level by a method we developed, in which mRNA molecules are labeled with fluorescent oligonucleotide probes. Transcription proceeded on almost all of the DNA molecules with a coiled conformation in the aqueous phase. In the presence of a tetravalent amine, spermine, the DNA had a folded conformation, and transcription was completely inhibited. When the Mg2+ concentration was increased, DNA was adsorbed onto the inner surface of the membrane and exhibited an extended conformation. The transcription experiments showed that this conformational transition recovered transcriptional activity; transcription occurred on DNA molecules that were on the membrane.

Singly and doubly labeled base-discriminating fluorescent oligonucleotide probes containing oxo-pyrene chromophore

We have developed new oxo-pyrene labeled fluorescent nucleoside, Oxo-PyU which showed a strong fluorescence dependency on solvent polarity at long wavelength. The designed singly and doubly Oxo-PyU labeled fluorescent oligonucleotide probes were found highly efficient for the discrimination of A and consecutive AA bases of target DNA opposite to the labeled base via generation of enhanced fluorescence signal.

Real‐Time Monitoring of RNA Synthesis in a Phospholipid‐Coated Microdroplet as a Live‐Cell Model

We constructed a phospholipid-coated water-in-oil microdroplet (20-60 microm in diameter) that encapsulated plasmid DNA containing a human beta-actin cDNA sequence, fluorescent oligonucleotide probes, and other components of the transcription reaction. Transcription inside individual microdroplets was investigated in real time by using fluorescence microscopy. The progress of the transcription reaction was successively monitored by fluorescence resonance energy transfer (FRET), which was derived from the specific hybridization of fluorescent oligonucleotide probes to the beta-actin mRNAs synthesized. In microdroplets composed of phosphatidylethanolamine, DNAs were located in the aqueous phase or on the membrane surface depending on the Mg(II) concentration. FRET images showed that transcription occurred on DNA in both states. Moreover, individual DNA molecules undergoing transcription were visualized as discrete FRET signals in cases in which small numbers of DNAs were present in the microdroplet.

Fluorescent oligonucleotide probe based on G-quadruplex scaffold for signal-on ultrasensitive protein assay

This work reported the G-quadruplex structure of pyrene-labeled G-rich DNA probe and its application in the immunoglobulin E (IgE) detection, providing plausibly an insight into the biological function of human telomere. Based on the intermolecular G-quadruplex, a terminal-single-pyrene-labeled oligonucleotide signaling probe was developed and a novel protein assay strategy was proposed via combining specific DNA cleavage by S1 nuclease with target-recognizing aptamer. This assay platform not only circumvented the optimization of specific sites for reporter attachment and the pyrene monomer fluorescence quenching by flanking nucleotide bases but also presented a signal-on mechanism. Thus, ultrasensitive homogeneous detection of IgE was successfully conducted. A linear dynamic range of 4.72 × 10 −12 to 7.56 × 10 −9 m, a regression coefficient of 0.9941 and a detection limit of 9.45 × 10 −14 m were given. Additionally, a preliminary concept of the single-pyrene-labeled excimer fluorescence probes associated with G-quadruplex for screening biological markers was described. Importantly, the unexpected structural property of G-quadruplex discovered seems to provide valuable information to allow understanding of the structure and function of human telomere and exploring of useful structure-based anticancer drug.

Design of extremely facile 3'- and 5'- ends free molecular beacons using C8 alkylamino substituted 2'-deoxyguanosine

A novel fluorescent oligonucleotide probe containing pyrene-labeled C8 alkylamino-substituted 2'-deoxyguanosine and a practically useful 3'- and 5'- ends-free self-quenched molecular beacons (MB) were designed. A unique MB detectable by pyrene excimer fluorescence was also demonstrated.

5183 Sequence variants in BRCA1 and BRCA2 genes detected by high-resolution melting analysis as a tool in molecular genetics analysis of inherited breast cancers

Current forensic DNA profiling methods rely on the analysis of samples at specialised laboratories with an average turnaround time of several days. The ability to rapidly determine a partial profile of short tandem repeats at the point-of-arrest would be of great benefit to police forces around the world, for example enabling a suspect to be rapidly included or excluded from an investigation. We have developed a homogeneous PCR method for the interrogation of STR loci utilising fluorescent oligonucleotide probes and melting curve analysis. Alleles of the D18S51, TH01 and D8S1179 loci were differentiated and identified on the basis of target length and probe melting temperature. Assay performance was evaluated by comparing melting peak data with the AmpFlSTR® SGM Plus® system. The method is compatible with direct analysis of unpurified buccal swab samples, enabling a partial STR profile to be generated within 1 h.

PCR-based DGGE and FISH analysis of methanogens in an anaerobic closed digester tank for treating palm oil mill effluent

16S ribosomal RNA (rRNA)-targeted fluorescent in situ hybridization combined with polymerase chain reaction (PCR)-cloning, light microscopy using Gram stains, scanning electron microscopy and denatured gradient gel electrophoresis were used to reveal the distribution of methanogens within an anaerobic closed digester tank fed with palm oil mill effluent. For specific detection of methanogens, 16S rRNA-cloning analysis was conducted followed by restriction fragment length polymorphism (RFLP) for presumptive identification of methanogens. To cover the drawbacks of the PCR-cloning study, the organization of the microorganisms was visualized in the activated sludge sample by using fluorescent oligonucleotide probes specific to several different methanogens, and a probe for bacteria. In situ hybridization with methanogens and bacterial probes and denatured gradient gel electrophoresis within activated sludge clearly confirmed the presence of Methanosaeta sp. and Methanosarcina sp. cells. Methanosaeta concilii was found to be the dominant species in the bioreactor. These results revealed the presence of possibly new strain of Methanosaeta in the bioreactor for treating palm oil mill effluent called Methanosaeta concilii SamaliEB (Gene bank accession number: EU580025). In addition, fluorescent hybridization pictured the close association between the methanogens and bacteria and that the number of methanogens was greater than the number of bacteria.

Validation of iQ-Check E. coli O157:H7 Real-Time PCR Test Kit for Detection of Escherichia coli O157:H7 in Selected Foods

iQ-Check E. coli O157:H7 (Bio-Rad Laboratories, Hercules, CA) is a real-time PCR kit for detection of E. coli O157:H7 from selected foods. Specific fluorescent oligonucleotide probes are used to detect target DNA during the amplification, by hybridizing to the amplicons. These fluorescent probes are linked to a fluorophore which fluoresces only when hybridized to the target sequence. Three foods (ground beef, apple cider, fresh spinach) were selected to compare the performance of iQ-Check E. coli O157:H7 to the U.S. Department of Agriculture-Food Safety and Inspection Service Microbiology Laboratory Guidebook (MLG) reference method for ground beef and the U.S. Food and Drug Administration/Bacteriological Analytical Manual reference method for apple cider and fresh spinach. Three protocols were tested in this study: a shortened 8 h primary enrichment in buffered peptone water (BPW), a 24 h enrichment in BPW, and an enrichment in appropriate reference method enrichment broth. The iQ-Check E. coli O157:H7 method was able to identify more true/confirmed positive samples than the reference method. Inclusivity and exclusivity rates of the method were 100%. iQ-Check E. coli O157:H7 performed as expected when minor procedural variations were introduced, validating the ruggedness of the method. There was no significant difference observed in performance over the shelf life of the kit.

A new fluorescent oligonucleotide probe for in situ detection of s-triazine-degrading Rhodococcus wratislaviensis in contaminated groundwater and soil samples

A bacterial strain (FPA1) capable of using terbuthylazine, simazine, atrazine, 2-hydroxysimazine, deethylatrazine, isopropylamine or ethylamine as its sole carbon source was isolated from a shallow aquifer chronically contaminated with s-triazine herbicides. Based on its 16S rDNA sequence analysis, the strain FPA1 was identified as Rhodococcus wratislaviensis. The disappearance time of 50% of the initial terbuthylazine concentration in the presence of this strain (DT 50) was 62 days. This strain was also able to mineralise the [U-ring 14C] triazine-ring, albeit at a slow rate. A 16S rRNA target oligonucleotide probe (RhLu) was designed, and the FISH protocol was optimised, in order to detect R. wratislaviensis in s-triazine-contaminated sites. The RhLu probe gave a positive signal (expressed as % of total DAPI-positive cells) in both the groundwater (2.19 ± 0.41%) and soil (2.10 ± 0.96%) samples analysed. Using the RhLu probe, R. wratislaviensis can be readily detected, and its population dynamics can be easily monitored, in soil and in water ecosystems contaminated with s-triazine. To the best of our knowledge, this is the first report showing the isolation, from groundwater, of a bacterial strain able to degrade s-triazines.

Imaging individual mRNA molecules using multiple singly labeled probes

We describe a method for imaging individual mRNA molecules in fixed cells by probing each mRNA species with 48 or more short, singly labeled oligonucleotide probes. This makes each mRNA molecule visible as a computationally identifiable fluorescent spot via fluorescence microscopy. We demonstrate simultaneous detection of three mRNA species in single cells and mRNA detection in yeast, nematodes, fruit fly wing discs, mammalian cell lines and neurons.