Rapid and specific detection of PCR products using light-up probes.

Newly developed light-up probes offer an attractive tool for PCR product detection. The light-up probe, which consists of a thiazole orange derivative linked to a peptide nucleic acid oligomer, hybridizes specifically to complementary nucleic acids. Upon hybridization the thiazole orange moiety interacts with the nucleic acid bases and the probe becomes brightly fluorescent. This eliminates the need to separate bound from unbound probes and reduces the risk of cross contamination during sample handling. We demonstrate here the applicability of light-up probes in two different PCR assays, one directed towards the human beta-actin gene and the other towards the invA gene of Salmonella. The probes do not interfere with the PCR reaction and can either be included in the sample mixture or added after completed amplification. The specificity of the probe is found to be excellent: a single-base mismatch in the target sequence is sufficient to prevent probe binding as indicated by the lack of fluorescence increase. Furthermore, a clear correlation is found between the intensity of gel bands and the measured probe fluorescence in solution, which suggests that the amount of PCR products can be quantified using light-up probes.

Interactions of the antiviral quinoxaline derivative 9-OH-B220 [2, 3-dimethyl-6-(dimethylaminoethyl)- 9-hydroxy-6H-indolo-[2, 3-b]quinoxaline] with duplex and triplex forms of synthetic DNA and RNA.

The binding of an antiviral quinoxaline derivative, 2,3-dimethyl- 6 - (dimethylaminoethyl) - 9 - hydroxy - 6H - indolo - [2,3 - b]quinoxaline (9-OH-B220), to synthetic double and triple helical DNA (poly(dA).poly(dT) and poly(dA).2poly(dT)) and RNA (poly(rA). poly(rU) and poly (rA).2poly(rU)) has been characterized using flow linear dichroism (LD), circular dichroism (CD), fluorescence spectroscopy, and thermal denaturation. When either of the DNA structures or the RNA duplex serve as host polymers a strongly negative LD is displayed, consistent with intercalation of the chromophoric ring system between the base-pairs/triplets of the nucleic acid structures. Evidence for this geometry also includes weak induced CD signals and strong increments of the fluorescence emission intensities upon binding of the drug to each of these polymer structures. In agreement with intercalative binding, 9-OH-B220 is found to effectively enhance the thermal stability of both the double and triple helical states of DNA as well as the RNA duplex. In the case of poly(dA).2poly(dT), the drug provides an unusually large stabilization of its triple helical state; upon binding of 9-OH-B220 the triplex-to-duplex equilibrium is shifted towards higher temperature by 52.5 deg. C in a 10 mM sodium cacodylate buffer (pH 7.0) containing 100 mM NaCl and 1 mM EDTA. When triplex RNA serves as host structure, LD indicates that the average orientation angle between the drug chromophore plane and the helix axis of the triple helical RNA is only about 60 to 65 degrees. Moreover, the thermal stabilizing capability, as well as the fluorescence increment, CD inducing power and perturbations of the absorption envelope, of 9-OH-B220 in complex with the RNA triplex are all less pronounced than those observed for the complexes with DNA and duplex RNA. These features indicate binding of 9-OH-B220 in the wide and shallow minor groove of poly(rA).2poly(rU). Based on the present results, some implications for the applications of this low-toxic, antiviral and easily administered drug in an antigene strategy, as well as its potential use as an antiretroviral agent, are discussed.

Binding mode of porphyrins to poly[d(A-T)(2)] and poly[d(G-C)(2)].

We examined the binding geometry of Co-meso-tetrakis (N-methyl pyridinium-4-yl)porphyrin, Co-meso-tetrakis (N-n-butyl pyridinium-4-yl)porphyrin and their metal-free ligands to poly[d(A-T)(2)] and poly[d(G-C)(2)] by optical spectroscopic methods including absorption, circular and linear dichroism spectroscopy, and fluorescence energy transfer technique. Signs of an induced CD spectrum in the Soret band depend only on the nature of the DNA sequence; all porphyrins exhibit negative CD when bound to poly[d(G-C)(2)] and positive when bound to poly[d(A-T)(2)]. Close analysis of the linear dichroism result reveals that all porphyrins exhibit outside binding when complexed with poly[d(A-T)(2)], regardless of the existence of a central metal and side chain. However, in the case of poly[d(G-C)(2)], we observed intercalative binding mode for two nonmetalloporphyrins and an outside binding mode for metalloporphyrins. The nature of the outside binding modes of the porphyrins, when complexed with poly[d(A-T)(2)] and poly[d(G-C)(2)], are quite different. We also demonstrate that an energy transfer from the excited nucleo-bases to porphyrins can occur for metalloporphyrins.

Unique base-pair breathing dynamics in PNA-DNA hybrids.

Kinetic and thermodynamic parameters, derived from 1H-NMR measurements of the imino proton exchange rates upon titration with the exchange catalyst ammonia, are reported for two mixed-sequence peptide nucleic acid (PNA)-DNA hybrids and their counterpart DNA duplex. The exchange times of the imino protons in the PNA strands extrapolate to very short base-pair lifetimes in the limit of infinite exchange catalyst concentration. This is not due to generally less stable base-pairs in PNA-DNA hybrids, since the lifetimes, apparent dissociation constants and thermodynamic stability (DeltaG degrees ) of the innermost DNA guanine imino protons are similar in the hybrid duplexes and in the DNA duplex. In addition, the apparent dissociation constants determined for PNA bases of the hybrids are of the same order as those of the corresponding bases in the DNA duplex. An exchange process from the closed state was found to be inconsistent with the experimental data. From these results, we conclude that opening and closing rates of the PNA guanine and thymine bases are at least two orders of magnitude higher than those of the corresponding bases in the DNA duplex. Unusual kinetics in the hybrids is also evident from the destabilization of the complementary DNA strand thymine bases, which exhibit base-pair dissociation constants increased by approximately two orders of magnitude compared to what is observed in the DNA duplex, while the DNA strand guanine bases are largely unaffected. The general pattern of the base-pair dynamics in the hybrids obtained when using trimethylamine as an exchange catalyst is the same as when using ammonia. However, the long base-pair lifetimes i. e. those of the DNA duplex and the guanine bases of the DNA strands in the hybrids, are approximately three to five times longer than when using ammonia. Thus, all opening events sensed by ammonia are not accessible to trimethylamine. These observations are discussed in regard to the mechanism of base-pair opening and the nature of the open state.

Effects of minor and major groove-binding drugs and intercalators on the DNA association of minor groove-binding proteins RecA and deoxyribonuclease I detected by flow linear dichroism.

Linear and circular dichroic spectroscopies have been employed to investigate the effects of small DNA ligands on the interactions of two proteins which bind to the minor groove of DNA, viz. RecA protein from Escherichia coli and deoxyribonuclease I (bovine pancreas). Ligands representing three specific non-covalent binding modes were investigated: 4',6-diamidino-2-phenylindole and distamycin A (minor groove binders), methyl green (major groove binder), and methylene blue, ethidium bromide and ethidium dimer (intercalators). Linear dichroism was demonstrated to be an excellent detector, in real time, of DNA double-strand cleavage by deoxyribonuclease I. Ligands bound in all three modes interfered with the deoxyribonuclease I digestion of dsDNA, although the level of interference varied in a manner which could be related to the ligand binding site, the ligand charge appearing to be less important. In particular, the retardation of deoxyribonuclease I cleavage by the major groove binder methyl green demonstrates that accessibility to the minor groove can be affected by occupancy of the opposite groove. Binding of all three types of ligand also had marked effects on the interaction of RecA with dsDNA in the presence of non-hydrolyzable cofactor adenosine 5'-O-3-thiotriphosphate, decreasing the association rate to varying extents but with the strongest effects from ligands having some minor groove occupancy. Finally, each ligand was displaced from its DNA binding site upon completion of RecA association, again demonstrating that modification of either groove can affect the properties and behaviour of the other. The conclusions are discussed against the background of previous work on the use of small DNA ligands to probe DNA-protein interactions.

DNA structural features responsible for sequence-dependent binding geometries of Hoechst 33258.

The complexes of Hoechst 33258 with poly[d(A-T)2], poly[d(I-C)2], and poly[d(G-C)2], and poly[d(G-m5C)2] were studied using linear dichroism, CD, and fluorescence spectroscopies. The Hoechst-poly[d(I-C)2] complex, in which there is no guanine amino group protruding in the minor groove, exhibits spectroscopic properties that are very similar to those of the Hoechst-poly[d(A-T)2] complex. When bound to both of these polynucleotides, Hoechst exhibits an average orientation angle of near 45 degrees relative to the DNA helix axis for the long-axis polarized low-energy transition, a relatively strong positive induced CD, and a strong increase in fluorescence intensity--leading us to conclude that this molecule also binds in the minor groove of poly[d(I-C)2]. By contrast, when bound to poly[d(G-C)2] and poly[d(G-m5C)2], Hoechst shows a distinctively different behavior. The strongly negative reduced linear dichroism in the ligand absorption region is consistent with a model in which part of the Hoechst chromophore is intercalculated between DNA bases. From the low drug:base ratio onset of excitonic effects in the CD and fluorescence emission spectra, it is inferred that another part of the Hoechst molecule may sit in the major groove of poly[d(G-C)2] and poly[d(G-m5C)2] and preferentially stacks into dimers, though this tendency is strongly reduced for the latter polynucleotide. Based on these results, the importance of the interactions of Hoechst with the exocyclic amino group of guanine and the methyl group of cytosine in determining the binding modes are discussed.

The interaction of ellipticine derivatives with nucleic acids studied by optical and 1H-nmr spectroscopy: effect of size of the heterocyclic ring system.

The DNA interaction of derivatives of ellipticine with heterocyclic ring systems with three, four, or five rings and a dimethylaminoethyl side chain was studied. Optical spectroscopy of drug complexes with calf thymus DNA, poly[(dA-dT).(dA-dT)], or poly[(dG-dC).(dG-dC)] showed a 10 nm bathochromic shift of the light absorption bands of the pentacyclic and tetracyclic compounds upon binding to the nucleic acids, which indicates binding by intercalation. For the tricyclic compound a smaller shift of 1-3 nm was observed upon binding to the nucleic acids. Flow linear dichroism studies show that the geometry of all complexes is consistent with intercalation of the ring system, except for the DNA and poly[(dG-dC).(dG-dC)] complexes of the tricyclic compound, where the average angle between the drug molecular plane and the DNA helix axis was found to be 65 degrees. One-dimensional 1H-nmr spectroscopy was used to study complexes between d(CGCGATCGCG)2 and the tricyclic and pentacyclic compounds. The results on the pentacyclic compound show nonselective broadening due to intermediate chemical exchange of most oligonucleotide resonances upon drug binding. The imino proton resonances are in slow chemical exchange, and new resonances with upfield shifts approaching 1 ppm appear upon drug binding, which supports intercalative binding of the pentacyclic compound. The results on the tricyclic compound show more rapid binding kinetics and very selective broadening of resonances. The data suggest that the tricyclic compound is in an equilibrium between intercalation and minor groove binding, with a preference to bind close to the AT base pairs with the side chain residing in the minor groove.

Interaction of cationic porphyrins with DNA.

Utilizing linear dichroism (LD), circular dichroism (CD), and fluorescence energy transfer, the binding geometries of a series of Co(3+)-porphyrins and their free ligands were examined. The compounds studied were Co-meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (CoTMPyP) and its free ligand (H2-TMPyP), Co-meso-tetrakis(N-n-butylpyridinium-4-yl)porphyrin (CoTBPyP) and its free ligand (H2TBPyP), and Co-meso-tetrakis(N-n-octylpyridinium-4-yl)porphyrin (CoTOPyP). The two non-metalloporphyrins exhibit negative LD, having angles of roughly 75 degrees relative to the DNA helix axis. They also display negative CD and a significant contact energy transfer from the DNA bases. On the other hand, the three metalloporphyrins display orientation angles of roughly 45 degrees between the porphyrin plane and the helix axis of DNA. Furthermore, they exhibit positive CD and no contact energy transfer from DNA bases. These observations show that the metalloporphyrins are not intercalated whereas non-metalloporphyrins having four freely rotating meso-aryl groups intercalate between the base pairs of DNA. In the presence of KHSO5, the cobalt porphyrins cleave closed circular PM2 DNA in a single strand manner, i.e., a single activation event on the porphyrin leads to a break in one of the DNA strands. A kinetic analysis of the cleavage data revealed that cleavage rates are in the order CoTMPyP > CoTBPyP > CoTOPyP with the difference being due to different DNA affinities rather than differences in cleavage rate-constants. Based on these and earlier observations, the metalloporphyrins appear bound to a partially melted region of DNA.