Cyanine dye dUTP analogs for enzymatic labeling of DNA probes.

Fluorescence in situ hybridization (FISH) has become and indispensable tool in a variety of areas of research and clinical diagnostics. Many applications demand an approach for simultaneous detection of multiple target sequences that is rapid and simple, yet sensitive. In this work, we describe the synthesis of two new cyanine dye-labeled dUTP analogs, Cy3-dUTP and Cy5-dUTP. They are efficient substrates for DNA polymerases and can be incorporated into DNA probes by standard nick translation, random priming and polymerase chain reactions. Optimal labeling conditions have been identified which yield probes with 20-40 dyes per kilobase. The directly labeled DNA probes obtained with these analogs offer a simple approach for multicolor multisequence analysis that requires no secondary detection reagents and steps.     

Synthesis and enzymatic incorporation of photolabile dUTP analogues into DNA and their applications for DNA labeling

Two novel photolabile nucleotide triphosphate (NTP) analogues were synthesized through Sonogashira coupling and their enzymatic incorporation into DNA was evaluated with three different DNA polymerases (Taq, Vent exo- and T4) by polymerase chain reaction. Both nucleotide triphosphate analogues were recognized by these DNA polymerases as substrates for primer extension. Light irradiation of PCR products removed the photolabile group and released the amino and carboxyl moieties. Further site-specific dual-labeling for oligodeoxynucleotides (ODNs) and random labeling for a long DNA construct with fluorophores were successfully achieved with incorporation of the photolabile amine modified deoxyuridine triphosphate (dUnTP).     

Ferrocene conjugated oligonucleotide for electrochemical detection of DNA base mismatch

We describe the synthesis, binding, and electrochemical properties of ferrocene-conjugated oligonucleotides (Fc-oligos). The key step for the preparation of Fc-oligos contains the coupling of vinylferrocene to 5-iododeoxyuridine via Heck reaction. The Fc-conjugated deoxyuridine phosphoramidite was used in the Fc-oligonucleotide synthesis. We show that thiol-modified Fc-oligos deposited onto gold electrodes possess potential ability in electrochemical detection of DNA base mismatch.     

Sequence-specific covalent labelling of DNA

Sequence-specific DNA modification is of significance for applications in bio- and nano-technology, medical diagnostics and fundamental life sciences research. Preferentially, labelling should be performed covalently, which avoids doubts about label dissociation from the DNA under various conditions. Several methods to label native DNA have been developed in the last two decades. Triple-helix-forming oligodeoxynucleotides and hairpin polyamides that bind DNA sequences specifically in the major and minor groove respectively were used as targeting devices for subsequent covalent labelling. In addition, enzyme-directed labelling approaches utilizing nicking endonucleases in combination with DNA polymerases or DNA methyltransferases have been employed. This review summarizes various techniques useful for functionalization of long native DNA.     

Modified linear polyethylenimine-cholesterol conjugates for DNA complexation

Linear polyethylenimine (LPEI) is an effective nonviral gene carrier with transfection levels equal or above branched polyethylenimine (BPEI) and exhibits a lower cytotoxicity profile than BPEI. High molecular weight LPEI M(w) 25 k was modified with cholesterol in three different geometries: linear shaped (L), T-shaped (T), and a combined linear/T-shaped (LT) forming the LPEI-cholesterol (LPC) conjugates LPC-L, LPC-T, and LPC-LT, respectively. Physical characterization of LPC/pDNA complexes included particle size, zeta potential, DNase protection, mIL-12 p70 expression, and cytotoxicity. The particle size was further confirmed by atomic force microscopy (AFM). The LPC-T/pDNA complexes were optimal at N/P 10/1 that resulted in a particle size of approximately 250 nm, which was confirmed by AFM, and a surface charge of +10 mV. These complexes also effectively protected the pDNA for up to 180 min in the presence of DNase I. B16-F0 cells transfected with LPC-L and LPC-T showed protein expression levels higher than LPEI alone and twice that of BPEI but without any significant loss in cell viability. These results were confirmed with EGFP flow cytometry and transfection of Renca cells. The differences in rates of transfection of the LPC/pDNA complexes is due in part to conformational changes from the point of complex formation to interaction with the plasma membrane. These conformation changes provide protection for unprotonated secondary amines in the LPEI backbone by hydrophobic protection of the cholesterol moiety that we termed "unprotonated reserves". Finally, we show that LPC conjugates exploit receptor-mediated endocytosis via the LDL-R pathway with transgene expression levels decreasing nearly 20% after saturating the LDL-R sites on MCF-7 cells with hLDL-R-Ab.     

Stem-loop oligonucleotides as tools for labelling double-stranded DNA

We report on a sequence-specific double-stranded DNA labelling strategy in which a stem-loop triplex forming oligonucleotide (TFO) is able to encircle its DNA target. Ligation of this TFO to either a short hairpin oligonucleotide or a long double-stranded DNA fragment leads to the formation of a topological complex. This process requires the hybridization of both extremities of the TFO to each other on a few base pairs. The effects of different factors on the formation of these complexes have been investigated. Efficient complex formation was observed using both GT or TC TFOs. The stem-loop structure enhances the specificity of the complex. The topologically linked TFO remains associated with its target even under conditions that do not favour triple-helix formation. This approach is sufficiently sensitive for detection of a 20-bp target sequence at the subfemtomolar level. This study provides new insights into the mechanics and properties of stem-loop TFOs and their complexes with double-stranded DNA targets. It emphasizes the interest of such molecules in the development of new tools for the specific labelling of short DNA sequences.     

Ferrocene-oligonucleotide conjugates for electrochemical probing of DNA.

Toward the development of a universal, sensitive and convenient method of DNA (or RNA) detection, electrochemically active oligonucleotides were prepared by covalent linkage of a ferrocenyl group to the 5'-aminohexyl-terminated synthetic oligonucleotides. Using these electrochemically active probes, we have been able to demonstrate the detection of DNA and RNA at femtomole levels by HPLC equipped with an ordinary electrochemical detector (ECD) [Takenaka,S., Uto,Y., Kondo,H., Ihara,T. and Takagi,M. (1994) Anal. Biochem., 218, 436-443]. Thermodynamic and electrochemical studies of the interaction between the probes and the targets are presented here. The thermodynamics obtained revealed that the conjugation stabilizes the triple-helix complexes by 2-3 kcal mol-1 (1-2 orders increment in binding constant) at 298 K, which corresponds to the effect of elongation of additional several base triplets. The main cause of this thermodynamic stabilization by the conjugation is likely to be the overall conformational change of whole structure of the conjugate rather than the additional local interaction. The redox potential of the probe was independent of the target structure, which is either single- or double stranded. However, the potential is slightly dependent (with a 10-30 mV negative shift on complexation) on the extra sequence in the target, probably because the individual sequence is capable of contacting or interacting with the ferrocenyl group in a slightly different way from each other. This small potential shift itself, however, does not cause any inconvenience on practical applications in detecting the probes by using ECD. These results lead to the conclusion that the redox-active probes are very useful for the microanalysis of nucleic acids due to the stability of the complexes, high detection sensitivity and wide applicability to the target structures (DNA and RNA; single- and double strands) and the sequences.     

Trifluoromethyldiazirine-Containing dUTP: Synthesis and Application in DNA/Protein Crosslinking

Abstract

The 5-[N-(4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzoyl)-3-aminoallyl]-2′-deoxyuridine-5′-triphosphate was synthesized via acylation of 5-aminoallyl-2′-deoxyuridine-5′-triphosphate with 4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzoate N-hydroxysuccinimide. It was used for the preparation of 30 bp ATFMD-DNA coding for promoter sequence. UV-Irradiation (365 nm) of the specific complex of this duplex and E. coli RNA polymerase leads to the effective crosslinking DNA with all protein subunits.     

Detection in situ of gamma-ray-induced DNA strand breaks in single cells: enzymatic labelling of free 3'-OH ends

We report a procedure allowing the detection and counting of free 3'-OH DNA strand extremities in single cells in situ. Terminal transferase (TdT) catalysed the incorporation of 3H-dGMP into fixed nuclei of human colonic adenocarcinoma cells (HT29), using free 3'-OH ends as initiator. Radioactivity was detected by autoradiography and determined quantitatively with a rapid image-processing system for grain counting. The initiator activity for TdT increases with the dose of gamma-rays in the dose range 2.5-20 Gy.     

DNA cross-linking with metallointercalator-peptide conjugates

Short peptides have been tethered to a DNA-intercalating ruthenium complex to create a photoactivated cross-linking reagent. The ruthenium complex, [Ru(phen)(bpy')(dppz)]2+ (phen = 1,10-phenanthroline, bpy' = 4-(butyric acid)-4'-methyl-2,2'-bipyridine, and dppz = dipyridophenazine), delivers the peptide to DNA and initiates the cross-linking reaction by oxidizing DNA upon irradiation in the presence of an oxidative quencher. The tethered peptide, only five to six residues in length, forms cross-links with the oxidized site in DNA. Cross-linking was detected and studied by gel electrophoresis and through spectroscopic measurements. The ruthenium-peptide complex is luminescent when bound to DNA, and the binding constants for several intercalator-peptide conjugates were determined by luminescence titration. The composition of the peptide affects both binding affinity and the extent of cross-linking. The greatest amounts of cross-linking were observed with tethered peptides that contained positively charged residues, either lysine or arginine. To test the impact of individual residues on cross-linking, the central residue in a 5-mer peptide was substituted with seven different amino acids. Though mutation of this position had only a small effect on the extent of cross-linking, it was discovered that peptides containing Trp or Tyr gave a distinctive pattern of products in gels. In experiments using the untethered peptide and ruthenium complex, it was determined that delivery of the peptide by the ruthenium intercalator is not essential for cross-linking; peptide attachment to the metal complex can constrain cross-linking. Importantly, the cross-linking adducts produced with ruthenium-peptide conjugates are luminescent and thus provide a luminescent cross-linking probe for DNA.     

Optimization of conditions for the enzymatic hydrolysis of phytoestrogen conjugates in urine and plasma

Optimal pH, temperature, and concentration of enzyme conditions for the rate of hydrolysis of five isoflavone conjugates (daidzein, O-desmethylangolensin, equol, genistein, and glycitein) and two lignans (enterodiol and enterolactone) from two biological matrices (urine and plasma) were studied using beta-glucuronidase from Helix pomatia. In addition, the use of mixtures of beta-glucuronidase and sulfatase enzymes from different sources was investigated to find enzyme preparations that contained lower amounts of naturally present phytoestrogens. Quantification of aglycones spiked with (13)C(3)-labeled internal standards was carried out by LC-MS/MS. In urine, all of the phytoestrogen conjugates hydrolyzed within 2h under standard hydrolysis conditions (24mul H. pomatia, pH 5, 37 degrees C). Hydrolysis rates were improved at 45 degrees C and by doubling the enzyme concentration and may be used to further reduce hydrolysis times down to 100min. In plasma, a 16-h hydrolysis was required to ensure complete hydrolysis of all conjugates. As with urine, the use of increased temperature or increased enzyme concentration reduced hydrolysis times for most analytes. However, the rate of hydrolysis in plasma was significantly slower than that in urine for all analytes except enterodiol, for which the reverse was true. Neither increased temperature nor increased enzyme concentration increased the rate of hydrolysis of enterolactone. Hydrolysis at pH 6 proved to be detrimental to hydrolysis of phytoestrogen conjugates, especially those in plasma. Other enzyme preparations from different sources, such as beta-glucuronidase from Escherichia coli, were found to contain lower amounts of contaminating phytoestrogens and showed increased enzyme activity for isoflavones, but lower activity for lignans, when used with other sulfatase enzymes. In addition, this involved complicating the analytical procedure through using mixtures of enzymes. Therefore, the use of beta-glucuronidase from H. pomatia combined with an enzyme "blank" to correct for phytoestrogen contamination was shown to be a suitable method for hydrolysis of phytoestrogens.     

Fidelity of enzymatic ligation for DNA computing.

We describe a convenient assay for rapid qualitative evaluation of hybridization/ligation fidelity. The approach uses randomized probe strands of DNA and restriction enzyme digestion after amplification of reaction products by the polymerase chain reaction (PCR). We report ligation efficiencies and fidelities of two DNA ligases, T4 DNA ligase and Thermus aquaticus (Taq) DNA ligase, over a range of temperatures.     

Specific labelling of replicating SPP1 DNA

Summary Specific labelling of replicating bacteriophage SPP1 DNA can be achieved by infection at nonpermissive temperature of a B. subtilis strain carrying the initation mutation dnaB ts134. Under these conditions host DNA synthesis is reduced by 90 to 95%. This technique was used to identify cistrons of SPP1 involved in phage DNA synthesis and to define intermediates in SPP1 replication.Experiments reported were part of the Doctoral Thesis submitted by K. Burger to the Freie Universität Berlin     

Differential labelling of DNA in higher plants

Under our experimental conditions labelling of DNA in higher plants with ³²P phosphate, ¹⁴C uridine and ¹⁴C thymidine shows two distinct species of labelled DNA: bulk-DNA and a satellite DNA. The bulk DNA (? = 1.696 g.cm⁻³) does not incorporate ³²P phosphate, but ¹⁴C thymidine. On the other hand the satellite-DNA (? = 1.720 g.cm⁻³) does not incorporate ¹⁴C thymidine, but ³²P phosphate. Both have ¹⁴C-Uridine as a precursor. An attempt has been made to establish the cellular localisation of these two DNA's.     

Coupling of redox indicator dyes into an enzymatic reaction cycle

The spectral properties of ten redox indicator dyes were evaluated with the aim of finding the optimal choice for coupling to enzymatic reactions with high sensitivity for the production of the reduced form. Eight of the dyes were selected for coupling into a reaction cycle formed by yeast alcohol dehydrogenase with substrates ethanol and nicotinamide adenine dinucleotide (NAD+) and diaphorase with substrates reduced nicotinamide adenine dinucleotide (NADH, produced by the prior reaction) and the oxidized form of the respective dye. Two of the dyes exhibited decreased absorption on reduction, whereas all (eight) tetrazolium dyes increased in their absorption substantially upon reduction. Bis-tetrazolium dyes had a significantly higher molar extinction coefficient (up to 23,000 M-1.cm-1) than mono-tetrazolium dyes (down to 8000 M-1.cm-1). Kinetically, most dyes could be reduced with NADH (and diaphorase), but the rate of reduction varied considerably among the dyes with nitroblue tetrazolium (NBT) and tetranitroblue tetrazolium (TNBT) being the fastest. Therefore, NBT and TNBT seem to be the most suitable for fast response.     

Microwave-mediated enzymatic modifications of DNA

Here we report microwave-induced specific cleavage, ligation, dephosphorylation, and phosphorylation of nucleic acids catalyzed by restriction endonucleases, T4 DNA ligase, T4 polynucleotide kinase, and calf intestinal alkaline phosphatase. The microwave-mediated method has dramatically reduced the reaction time to 20 to 50 s. In control experiments, the same reactions failed to give the desired reaction products when carried out in the same time periods but without microwave irradiation. Because the microwave method is rapid, it could be a useful alternative to the time-consuming conventional procedure for enzymatic modification of DNA.     

Detection of DNA Damage in Prokaryotes by Terminal Deoxyribonucleotide Transferase-Mediated dUTP Nick End Labeling

Numerous agents can damage the DNA of prokaryotes in the environment (e.g., reactive oxygen species, irradiation, and secondary metabolites such as antibiotics, enzymes, starvation, etc.). The large number of potential DNA-damaging agents, as well as their diverse modes of action, precludes a simple test of DNA damage based on detection of nucleic acid breakdown products. In this study, free 3′-OH DNA ends, produced by either direct damage or excision DNA repair, were used to assess DNA damage. Terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick end labeling (TUNEL) is a procedure in which 3′-OH DNA ends are enzymatically labeled with dUTP-fluorescein isothiocyanate using TdT. Cells labeled by this method can be detected using fluorescence microscopy or flow cytometry. TUNEL was used to measure hydrogen peroxide-induced DNA damage in the archaeon Haloferax volcanii and the bacterium Escherichia coli. DNA repair systems were implicated in the hydrogen peroxide-dependent generation of 3′-OH DNA ends by the finding that the protein synthesis inhibitors chloramphenicol and diphtheria toxin blocked TUNEL labeling of E. coli and H. volcanii, respectively. DNA damage induced by UV light and bacteriophage infection was also measured using TUNEL. This methodology should be useful in applications where DNA damage and repair are of interest, including mutant screening and monitoring of DNA damage in the environment.