Minor groove binder-conjugated DNA probes for quantitative DNA detection by hybridization-triggered fluorescence

Here we describe the properties of a novel class of oligonucleotide probes capable of sensitive hybridization-triggered fluorescence. These fluorogenic probes, known commercially as MGB Eclipse probes, are characterized by having a conjugated minor groove binder (MGB) ligand at the 5'-end and a fluorophore at the 3'-end. Additionally, they have an efficient quencher moiety at the 5'-end that is useful with a wide variety of fluorescent dyes. Fluorescence of the single-stranded MGB Eclipse probe is efficiently quenched by the interaction of the terminal dye and quencher groups when not hybridized. Upon hybridization to a complementary target, the MGB molecule folds into duplex and hyper-stabilizes it, allowing the use of shorter, more specific probe sequences. The 5'-MGB-quencher group also prevents nuclease digestion by Taq DNA polymerase during PCR. Because of the hybridization-triggered fluorescence and the excellent specificity imparted by the MGB, these 5'-MGB Eclipse probes have great versatility for real-time PCR applications. The high sensitivity and specificity are illustrated using single nucleotide polymorphism detection, viral load determination, and gene expression analysis.     

Oligolabeling DNA probes to high specific activity with sequenase

In this protocol we present a reproducible method of preparing DNA probes of high specific activity using Sequenase. The probes produced by this method had a specific activity of 2.8×10⁹ cpm/μg with 69% of the total radioactivity incorporated into the TCA-precipitable materials. Probes with 5 to 10-fold lower specific activity were obtained using commercially available kits or using currently empolyed methods.     

Producing single-stranded DNA probes with the Taq DNA polymerase: a high yield protocol

We report an efficient procedure to synthesize either single- or double-stranded probes labeled with biotin-11-dUTP, biotin-21-dUTP or digoxigenin-11-dUTP. To produce the single-stranded probe, only a single primer is utilized in a Taq polymerase amplification of 55 cycles. A cytomegalovirus probe is presented. This procedure allows easy production of nonradioactively labeled pure single-stranded probes of any desired length and specificity.     

Novel gratisin derivatives with high antimicrobial activity and low hemolytic activity

The substitution of each constituent amino acid residue of gratisin (GR) with Ala residue indicated that each side chain structure of the constituent amino acid residues affect largely the antibiotic and hemolytic activities of GR. Among them, the substitution of Pro residues at positions 5 and 5′ with a cationic amino acid residues (Lys and Arg) results the high antibiotic activity and the low toxicity against human blood cells. Thus, we have found a novel position on the scaffold of GR at Pro^5,5′ residues whose modification will significantly lower the unwanted hemolytic activity and enhance the desired antibiotic activity.     

Stability, specificity and fluorescence brightness of multiply-labeled fluorescent DNA probes.

In this work, we studied the fluorescence and hybridization of multiply-labeled DNA probes which have the hydrophilic fluorophore 1-(straightepsilon-carboxypentynyl)-1'-ethyl- 3,3,3', 3'-tetramethylindocarbocyanine-5,5'-disulfonate (Cy3) attached via either a short or long linker at the C-5 position of deoxyuridine. We describe the effects of labeling density, fluorophore charge and linker length upon five properties of the probe: fluorescence intensity, the change in fluorescence upon duplex formation, the quantum yield of fluorescence (Phif), probe-target stability and specificity. For the hydrophilic dye Cy3, we have demonstrated that the fluorescence intensity andPhifare maximized when labeling every 6th base using the long linker. With a less hydrophilic dye, a labeling density this high could not be achieved without serious quenching of the fluorescence. The target specificity of multiply-labeled DNA probes was just as high as compared to the unmodified control probe, however, a less stable probe-target duplex is formed that exhibits a lower melting temperature. A mechanism that accounts for this destabilization is proposed which is consistent with our data. It involves dye-dye and dye-nucleotide interactions which appear to stabilize a single-stranded conformation of the probe.     

Novel fluorescence sensing methods for high throughput screening

We describe two new methods of fluorescence sensing for use in high throughput screening (HTS). Modulation sensing transforms analyte-dependent intensity changes into a change in the low-frequency modulation signal. Polarization sensing transforms an intensity change into a change in polarization. Both methods are internally calibrated by using a reference film immediately adjacent to the sample, which can be readily located on the HTS plate or on a nearby optical component and provides an intensity or polarization reference. Modulation sensing and polarization sensing were both shown useful for measurements of fluorophore concentrations, pH, or calcium concentrations in the wells of HTS plates. Sensing with a reference film provides the opportunity to internally reference HTS measurements without the need for additions to the sample. This approach can provide standardization for assays performed at different times.     

Conformation-sensitive nucleoside analogues as topology-specific fluorescence turn-on probes for DNA and RNA G-quadruplexes

Development of probes that can discriminate G-quadruplex (GQ) structures and indentify efficient GQ binders on the basis of topology and nucleic acid type is highly desired to advance GQ-directed therapeutic strategies. In this context, we describe the development of minimally perturbing and environment-sensitive pyrimidine nucleoside analogues, based on a 5-(benzofuran-2-yl)uracil core, as topology-specific fluorescence turn-on probes for human telomeric DNA and RNA GQs. The pyrimidine residues of one of the loop regions (TTA) of telomeric DNA and RNA GQ oligonucleotide (ON) sequences were replaced with 5-benzofuran-modified 2′-deoxyuridine and uridine analogues. Depending on the position of modification the fluorescent nucleoside analogues distinguish antiparallel, mixed parallel-antiparallel and parallel stranded DNA and RNA GQ topologies from corresponding duplexes with significant enhancement in fluorescence intensity and quantum yield. Further, these GQ sensors enabled the development of a simple fluorescence binding assay to quantify topology- and nucleic acid-specific binding of small molecule ligands to GQ structures. Together, our results demonstrate that these nucleoside analogues are useful GQ probes, which are anticipated to provide new opportunities to study and discover efficient G-quadruplex binders of therapeutic potential.     

Synthesis of probes with broad pH range fluorescence

Reaction of a rhodamine 2'-ester with an excess of alkyldiamines provides amino-functionalized rhodamine spirolactams, which when subsequently conjugated with carboxyfluorescein, provides probes which are fluorescent at acidic, neutral, and basic pH ranges.     

Fluorescence correlation spectroscopy with high count rate and low background: analysis of translational diffusion

An epi-illuminated microscope configuration for use in fluorescence correlation spectroscopy in bulk solutions has been analyzed. For determining the effective sample dimensions the spatial distribution of the molecule detection efficiency has been computed and conditions for achieving quasi-cylindrical sample shape have been derived. Model experiments on translational diffusion of rhodamine 6G have been carried out using strong focusing of the laser beam, small pinhole size and an avalanche photodiode in single photon counting mode as the detector. A considerable decrease in background light intensity and measurement time has been observed. The background light is 40 times weaker than the fluorescence signal from one molecule of Rh6G, and the correlation function with signal-to-noise ratio of 150 can be collected in 1 second. The effect of the shape of the sample volume on the autocorrelation function has been discussed. Correspondence to: R. Rigler     

Manufacturing DNA microarrays of high spot homogeneity and reduced background signal

Analyses on DNA microarrays depend considerably on spot quality and a low background signal of the glass support. By using betaine as an additive to a spotting solution made of saline sodium citrate, both the binding efficiency of spotted PCR products and the homogeneity of the DNA spots is improved significantly on aminated surfaces such as glass slides coated with the widely used poly-L-lysine or aminosilane. In addition, non-specific background signal is markedly diminished. Concomitantly, during the arraying procedure, the betaine reduces evaporation from the microtitre dish wells, which hold the PCR products. Subsequent blocking of the chip surface with succinic anhydride was improved considerably in the presence of the non-polar, non-aqueous solvent 1,2-dichloroethane and the acylating catalyst N:-methylimidazole. This procedure prevents the overall background signal that occurs with the frequently applied aqueous solvent 1-methyl-2-pyrrolidone in borate buffer because of DNA that re-dissolves from spots during the blocking process, only to bind again across the entire glass surface.     

Combinatorial labelling of DNA probes enables multicolour fluorescence in situ hybridisation in plants

This paper demonstrates a simple but effective use of combinatorial probes to label plant chromosomes by multicolour fluorescence in situ hybridisation (FISH). Three different DNA probes were labelled with only two different fluorophores, hybridised to somatic metaphase chromosomes of Secale cereale and Triticum aestivum, simultaneously visualised, and unequivocally distinguished in a single FISH experiment. Combinatorial labelling can augment karyotypical investigations, physical mapping of chromosomes and other analyses in plants based upon FISH.     

Sensitive fluorescence in situ hybridization signal detection in maize using directly labeled probes produced by high concentration DNA polymerase nick translation

The signal produced by fluorescence in situ hybridization (FISH) often is inconsistent among cells and sensitivity is low. Small DNA targets on the chromatin are difficult to detect. We report here an improved nick translation procedure for Texas red and Alexa Fluor 488 direct labeling of FISH probes. Brighter probes can be obtained by adding excess DNA polymerase I. Using such probes, a 30 kb yeast transgene, and the rp1, rp3 and zein multigene clusters were clearly detected.     

Sensitive fluorescence in situ hybridization signal detection in maize using directly labeled probes produced by high concentration DNA polymerase nick translation

The signal produced by fluorescence in situ hybridization (FISH) often is inconsistent among cells and sensitivity is low. Small DNA targets on the chromatin are difficult to detect. We report here an improved nick translation procedure for Texas red and Alexa Fluor 488 direct labeling of FISH probes. Brighter probes can be obtained by adding excess DNA polymerase I. Using such probes, a 30?kb yeast transgene, and the rp1, rp3 and zein multigene clusters were clearly detected.     

DNA probes using fluorescence resonance energy transfer (FRET): designs and applications.

Fluorescence resonance energy transfer (FRET) is widely used in biomedical research as a reporter method. Oligonucleotides with a DNA backbone and one or several chromophore tags have found multiple applications as FRET probes. They are especially advantageous for the real-time monitoring of biochemical reactions and in vivo studies. This paper reviews the design and applications of various DNA-based probes that use FRET The approaches used in the design of new DNA FRET probes are discussed.     

Molecular cloning of complementary DNA: preparation of a plasmid vector with low transformation background

A simple method that allows the rapid preparation of oligo dG-tailed plasmid vectors is presented. The procedure involves purification of the tailed molecules by hybridization to oligo dC-cellulose followed by a stepwise thermal elution. The resulting plasmid is virtually devoid of transformation activity in the absence of oligo dC-tailed DNA fragments. It allows construction of cDNA libraries with as low as 1% of colonies harboring wild-type plasmids.     

Sensitive fluorescence in situ hybridization signal detection in maize using directly labeled probes produced by high concentration DNA polymerase nick translation.

The signal produced by fluorescence in situ hybridization (FISH) often is inconsistent among cells and sensitivity is low. Small DNA targets on the chromatin are difficult to detect. We report here an improved nick translation procedure for Texas red and Alexa Fluor 488 direct labeling of FISH probes. Brighter probes can be obtained by adding excess DNA polymerase I. Using such probes, a 30 kb yeast transgene, and the rp1, rp3 and zein multigene clusters were clearly detected.     

Use of 2-aminopurine and tryptophan fluorescence as probes in kinetic analyses of DNA polymerase beta

Although the use of 2-aminopurine (2-AP) as a probe in stopped-flow analyses of DNA polymerase beta (Pol beta) had provided important mechanistic insight, the conditions used were limited by the location of 2-AP and the use of a combination of tryptophan (Trp) and 2-AP fluorescence. This study examined different DNA substrates to identify several factors that can affect the observed signal in stopped-flow experiments. Both Trp and 2-AP emissions were separately excited and monitored. It was found that both probes show a fast phase and a slow phase of fluorescence changes, but the direction and the amplitude vary greatly between the two probes and between different DNA substrates. Detailed analyses suggested that the location of 2-AP in the template has a significant impact on the fluorescence properties of 2-AP and that a location opposite the incoming dNTP, which has been used in all such studies in the past, is not optimal. In particular, the results show that placing 2-AP one base after the templating base greatly enhances the signal intensity, which suggests a significant change in base stacking interactions at this position during nucleotide incorporation. These results allowed us to derive an improved set of conditions which were then used to reevaluate results from previous reports. It also allows greater freedom in the type of base pairs studied, since 2-AP is not the templating base in the nascent base pair. Kinetic constants were determined for dNTP and catalytic Mg(2+). The results obtained from stopped-flow experiments were compared to results from chemical quench. Stopped flow of incorrect dNTP incorporation and the reverse reaction are also reported, which provide useful information to the mechanism of Pol beta.