OFF-to-ON type fluorescent probe for the detection of 8-oxo-dG in DNA by the Adap-masked ODN probe

We have recently reported that Adap (adenosine-1,3-diazaphenoxazine) is an artificial nucleoside analogue for the specific recognition by multiple hydrogen bonding and that its fluorescence is selectively quenched with 8-oxo-2'-deoxyguanosine (8-oxo-dG) in DNA. We now report the development of a new OFF-to-ON type FRET probe, in which one strand contains Adap and another contains natural nucleotides for the formation of a less stable double strand. Each strand was labeled with Cy3 or BHQ2 at the 5'-end or 3'-end, respectively. It was expected in this system that fluorescence of the duplex probe is first quenched by FRET, but the target DNA strand containing 8-oxo-dG at the complementary site of Adap would enhance the displacement reaction of the less stable duplex probe that results in the fluorescence recovery. The results showed that the duplex probe containing the Adap-T base pair exhibited a complete discrimination between 8-oxo-dG and dG in DNA by fluorescence enhancement.     

Effect of the bases flanking an abasic site on the recognition of nucleobase by amiloride

Background

We explain here the various non-covalent interactions which are responsible for the different binding modes of a small ligand with DNA.

Methods

The combination of experimental and theoretical methods was used.

Results

The interaction of amiloride with thymine was found to depend on the bases flanking the AP site and different binding modes were observed for different flanking bases. Molecular modeling, absorption studies and binding constant measurements support for the different binding patterns. The flanking base dependent recognition of AP site phosphates was investigated by ³¹P NMR experiments. The thermodynamics of the ligand–nucleotide interaction was demonstrated by isothermal titration calorimetry. The emission behavior of amiloride was found to depend on the bases flanking the AP site. Amiloride photophysics in the context of AP-site containing DNA is investigated by time-dependent density functional theory.

Conclusions

Flanking bases affect the ground and excited electronic states of amiloride when binding to AP site, which causes flanking base-dependent fluorescence signaling.

General significance

The various noncovalent interactions have been well characterized for the determination of nucleic acid structure and dynamics, and protein–DNA interactions. However, these are not clear for the DNA–small molecule interactions and we believe that our studies will bring a new insight into such phenomena.     

DNA Abasic Site-Selective Enhancement of Sanguinarine Fluorescence with a Large Emission Shift

Small molecules that can specifically bind to a DNA abasic site (AP site) have received much attention due to their importance in DNA lesion identification, drug discovery, and sensor design. Herein, the AP site binding behavior of sanguinarine (SG), a natural alkaloid, was investigated. In aqueous solution, SG has a short-wavelength alkanolamine emission band and a long-wavelength iminium emission band. At pH 8.3, SG experiences a fluorescence quenching for both bands upon binding to fully matched DNAs without the AP site, while the presence of the AP site induces a strong SG binding and the observed fluorescence enhancement for the iminium band are highly dependent on the nucleobases flanking the AP site, while the alkanolamine band is always quenched. The bases opposite the AP site also exert some modifications on the SG''s emission behavior. It was found that the observed quenching for DNAs with Gs and Cs flanking the AP site is most likely caused by electron transfer between the AP site-bound excited-state SG and the nearby Gs. However, the flanking As and Ts that are not easily oxidized favor the enhanced emission. This AP site-selective enhancement of SG fluorescence accompanies a band conversion in the dominate emission from the alkanolamine to iminium band thus with a large emission shift of about 170 nm. Absorption spectra, steady-state and transient-state fluorescence, DNA melting, and electrolyte experiments confirm that the AP site binding of SG occurs and the stacking interaction with the nearby base pairs is likely to prevent the converted SG iminium form from contacting with water that is thus emissive when the AP site neighbors are bases other than guanines. We expect that this fluorophore would be developed as a promising AP site binder having a large emission shift.     

Fluorescent aptasensors based on conformational adaptability of abasic site-containing aptamers in combination with abasic site-binding ligands

Aptamers are nucleic acids that can selectively bind to a variety of targets. Aptamers usually undergo conformational transitions from a flexible or disordered structure into a rigid or ordered structure upon target-binding. This study describes a detection method for l-argininamide (l-Arm) and adenosine based on the conformational adaptability of nucleic acid aptamers. An abasic site (AP site) was formed in the stem and close to the target-binding site of a stem-loop aptamer as an anchoring pocket for a fluorescent ligand. 3,5-Diamino-6-chloro-2-pyrazine carbonitrile (DCPC), which can bind to AP site-containing DNA duplexes by pseudo-base pairing, was utilized as a signaling reporter for the target-binding. The binding of a target to an aptamer induces the tight pairing of bases flanking the AP site, so that DCPC can effectively bind to the stem. The binding of DCPC is accompanied by a significant enhancement of its fluorescence. This new sensing method without an antisense DNA strand was demonstrated by using l-Arm and its aptamer as a model. It was confirmed that the method can sensitively detect l-Arm with a detection limit of 2.1 μM. The proposed method was also applied to adenosine detection, where the reported sequence of an adenosine aptamer was slightly modified. The method based on an AP site-containing aptamer and an AP site-binding ligand was applicable to detection of a target in horse serum.     

Using 2-aminopurine fluorescence to detect bacteriophage T4 DNA polymerase-DNA complexes that are important for primer extension and proofreading reactions

The fluorescence of the base analogue 2-aminopurine (2AP) was used to probe bacteriophage T4 DNA polymerase-induced conformational changes in the template strand produced during the nucleotide incorporation and proofreading reactions. 2AP fluorescence in DNA is quenched by 2AP interactions with neighboring bases, but T4 DNA polymerase binding to DNA substrates labeled with 2AP in the templating position produces large increases in fluorescence intensity. Fluorescence lifetime studies were performed to characterize the fluorescent complexes. Three fluorescence lifetime components were observed for unbound DNA substrates as reported previously, but T4 DNA polymerase binding modulated the amplitudes of these components and created a new, highly fluorescent 10.5 ns component. Experimental evidence for correlation of fluorescence lifetimes with functionally distinct complexes was obtained by forming complexes under different reaction conditions. T4 DNA polymerase complexes were formed with DNA substrates with matched and mismatched primer ends and with A+T- or G+C-rich primer-terminal regions. dTTP was added to binary complexes to form ternary DNA polymerase-DNA-nucleotide complexes. The effect of temperature on complex formation was studied, and complexes were formed with proofreading-defective T4 DNA polymerases. Complexes characterized by the 10.5 ns lifetime were demonstrated to be formed at the crossroads of the primer-extension and proofreading pathways.     

15-mer DNA duplexes containing an abasic site are thermodynamically more stable with adjacent purines than with pyrimidines

Abasic site (AP)-containing duplexes, with flanking adenine (A) or cytosine (C) bases, were shown to be more stable with flanking A than with C bases [Sági, J., Hang, B., and Singer, B. (1999) Chem. Res. Toxicol. 12, 917-923]. We investigated whether the lower-magnitude destabilization by an AP site, with A neighbors, is a general effect of the purine versus the pyrimidine neighbors. Duplex stability, as compared to that of the corresponding control duplexes, was markedly decreased by the incorporation of the AP site (x) opposite any of the four bases. However, for the duplexes containing T, A, or C opposite the AP site, replacement of the symmetric doublet flanking pyrimidine bases with purines resulted in a smaller destabilization effect. The average stabilizing effect of the symmetric doublet purine neighbors of an AP site opposite T, A, or C bases was 3.2 degrees C (DeltaT(m)) and 1.3 kcal/mol (DeltaDeltaG degrees (37)) compared to those of pyrimidine neighbors. In contrast, a G.AP pair reduced or eliminated the differential effect of the neighbors. Using unrestrained molecular dynamics, it was shown that for the duplexes containing T opposite the AP site, with doublet pyrimidine neighbors, there was a larger magnitude of curvature around the lesion site than for the duplexes with the purines flanking the AP site. Purines flanking the AP site tend to shift toward each other, creating overlap, in contrast to the flanking pyrimidines. This indicates the possibility of stacking between purine bases at the AP site and can be the reason for the observed smaller thermodynamic destabilization of the duplexes with the AAxAA and GGxGG central sequences, as compared to those with TTxTT and CCxCC sequences. This work showed that for an AP site the GC content is not the only determinant of duplex stability, but rather is influenced more by whether purines or pyrimidines flank the AP site.     

Dynamics of mismatched base pairs in DNA

The structural dynamics of mismatched base pairs in duplex DNA have been studied by time-resolved fluorescence anisotropy decay measurements on a series of duplex oligodeoxynucleotides of the general type d[CGG(AP)GGC].d[GCCXCCG], where AP is the fluorescent adenine analogue 2-aminopurine and X = T, A, G, or C. The anisotropy decay is caused by internal rotations of AP within the duplex, which occur on the picosecond time scale, and by overall rotational diffusion of the duplex. The correlation time and angular range of internal rotation of AP vary among the series of AP.X mismatches, showing that the native DNA bases differ in their ability to influence the motion of AP. These differences are correlated with the strength of base-pairing interactions in the various AP.X mismatches. The interactions are strongest with X = T or C. The ability to discern differences in the strength of base-pairing interactions at a specific site in DNA by observing their effect on the dynamics of base motion is a novel aspect of the present study. The extent of AP stacking within the duplex is also determined in this study since it influences the excited-state quenching of AP. AP is thus shown to be extrahelical in the AP.G mismatch. The association state of the AP-containing oligodeoxynucleotide strand is determined from the temperature-dependent tumbling correlation time. An oligodeoxynucleotide triplex is formed with a particular base sequence in a pH-dependent manner.     

Synthesis and binding properties of new selective ligands for the nucleobase opposite the AP site

DNA is continuously damaged by endogenous and exogenous factors such as oxidative stress or DNA alkylating agents. These damaged nucleobases are removed by DNA N-glycosylase and form apurinic/apyrimidinic sites (AP sites) as intermediates in the base excision repair (BER) pathway. AP sites are also representative DNA damages formed by spontaneous hydrolysis. The AP sites block DNA polymerase and a mismatch nucleobase is inserted opposite the AP sites by polymerization to cause acute toxicities and mutations. Thus, AP site specific compounds have attracted much attention for therapeutic and diagnostic purposes. In this study, we have developed nucleobase-polyamine conjugates as the AP site binding ligand by expecting that the nucleobase part would play a role in the specific recognition of the nucleobase opposite the AP site by the Watson-Crick base pair formation and that the polyamine part should contribute to the access of the ligand to the AP site by a non-specific interaction to the DNA phosphate backbone. The nucleobase conjugated with 3,3'-diaminodipropylamine (A-ligand, G-ligand, C-ligand, T-ligand and U-ligand) showed a specific stabilization of the duplex containing the AP site depending on the complementary combination with the nucleobase opposite the AP site; that is A-ligand to T, G-ligand to C, C-ligand to G, T- and U-ligand to A. The thermodynamic binding parameters clearly indicated that the specific stabilization is due to specific binding of the ligands to the complementary AP site. These results have suggested that the complementary base pairs of the Watson-Crick type are formed at the AP site.     

DNA damage by thiol-activated neocarzinostatin chromophore at bulged sites

Bulge structures in nucleic acids are of general biological significance and are potential targets for therapeutic drugs. It has been shown in a previous study that thiol-activated neocarzinostatin chromophore is able to cleave duplex DNA selectively at a position opposite a single unpaired cytosine or thymine base on the 3' side. In this work, we studied in greater detail the nature of this type of cleavage and the basis for the selectivity of the bulge site cleavage over the usual strand cleavage at a T site in the duplex region by using duplexes containing an internal control and a bulge, which is composed of different types and number of bases. Experimental results indicated that the bulge-induced cleavage is initiated by 5' hydrogen abstraction and is greatly affected by the base composition of the bulge. A single-base bulge, especially when containing a purine, yields higher efficiency and greater selectivity for the bulge-induced cleavage. In particular, a single adenine base gives rise to the highest cleavage yield and provides over 20 times greater selectivity for cleavage at the bulge site compared with the internal control site in duplexes. The binding dissociation constants of postactivated drug for a stem-loop structure containing a one- or two-base bulge in the stem, measured by fluorescence quenching, show that the binding is about 3-4 times stronger for bulge-containing duplexes than for perfect hairpin duplexes. For RNA.DNA hybrid duplexes, where the DNA is the target strand and the RNA is the bulge-containing strand, bulge-induced cleavage was observed, although at low yield. On the other hand, when RNA is the nonbulge strand, no bulge-induced cleavage was found. When the reaction is performed in the absence of oxygen, the major product is a covalent adduct, and it is at the same location as the cleavage site under aerobic conditions.     

Requirements for noncovalent binding of vaccinia topoisomerase I to duplex DNA.

Vaccinia DNA topoisomerase binds duplex DNA and forms a covalent adduct at sites containing a conserved sequence element 5'(C/T)CCTT decreases in the scissile strand. Distinctive aspects of noncovalent versus covalent interaction emerge from analysis of the binding properties of Topo(Phe-274), a mutated protein which is unable to cleave DNA, but which binds DNA noncovalently. Whereas DNA cleavage by wild type enzyme is most efficient with 'suicide' substrates containing fewer than 10 base pairs distal to the scissile bond, optimal noncovalent binding by Topo(Phe-274) requires at least 10-bp of DNA 3' of the cleavage site. Thus, the region of DNA flanking the pentamer motif serves to stabilize the noncovalent topoisomerase-DNA complex. This result is consistent with the downstream dimensions of the DNA binding site deduced from nuclease footprinting. Topo(Phe-274) binds to duplex DNA lacking the consensus pentamer with 7-10-fold lower affinity than to CCCTT-containing DNA.     

2-Aminopurine as a fluorescent probe for DNA base flipping by methyltransferases.

DNA base flipping, which was first observed for the C5-cytosine DNA methyltransferase M. Hha I, results in a complete removal of the stacking interactions between the target base and its neighbouring bases. We have investigated whether duplex oligodeoxynucleotides containing the fluorescent base analogue 2-aminopurine can be used to sense DNA base flipping. Using M. Hha I as a paradigm for a base flipping enzyme, we find that the fluorescence intensity of duplex oligodeoxynucleotides containing 2-aminopurine at the target site is dramatically enhanced (54-fold) in the presence of M. Hha I. Duplex oligodeoxynucleotides containing 2-aminopurine adjacent to the target cytosine show little fluorescence increase upon addition of M. Hha I. These results clearly demonstrate that duplex oligodeoxynucleotides containing 2-aminopurine at the target site can serve as fluorescence probes for base flipping. Another enzyme hypothesized to use a base flipping mechanism is the N6-adenine DNA methyltransferase M. Taq I. Addition of M. Taq I to duplex oligodeoxynucleotides bearing 2-aminopurine at the target position, also results in a strongly enhanced fluorescence (13-fold), whereas addition to duplex oligodeoxynucleotides containing 2-aminopurine at the 3'- or 5'-neighbouring position leads only to small fluorescence increases. These results give the first experimental evidence that the adenine-specific DNA methyltransferase M. Taq I also flips its target base.     

Efficiency of incision of an AP site within clustered DNA damage by the major human AP endonuclease.

A major DNA lesion induced by ionizing radiation and formed on removal of oxidized base lesions by various glycosylases is an apurinic/apyrimidinic site (AP site). The presence of an AP site within clustered DNA damage, induced following exposure to ionizing radiation or radiomimetic anticancer agents, may present a challenge to the repair machinery of the cell, if the major human AP endonuclease, HAP1, does not efficiently incise the AP site. In this study, specific oligonucleotide constructs containing an AP site located at several positions opposite to another damage [5,6-dihydrothymine (DHT), 8-oxoG, AP site, or various types of single strand breaks] on the complementary strand were used to determine the relative efficiency of the purified HAP1 protein in incising an AP site(s) from clustered DNA damage. A base damage (DHT and 8-oxoG) on the opposite strand has little or no influence on the rate of incision of an AP site by HAP1. In contrast, the presence of either a second AP site or various types of single strand breaks, when located one or three bases 3' to the base opposite to the AP site, has a strong inhibitory effect on the efficiency of incision of an AP site. The efficiency of binding of HAP1 to an AP site is reduced by approximately 1 order of magnitude if a single strand break (SSB) is located one or three bases 3' to the site opposite to the AP site on the complementary strand. If the AP site and either a SSB or a second AP site are located at any of the other positions relative to each other, a double strand break may result.     

Interaction of DNA polymerase I (Klenow fragment) with DNA substrates containing extrahelical bases: implications for proofreading of frameshift errors during DNA synthesis

Frameshift mutagenesis occurs through the misalignment of primer and template strands during DNA synthesis and involves DNA intermediates that contain one or more extrahelical bases in either strand of the DNA substrate. To investigate whether these DNA structures are recognized by the proofreading apparatus of DNA polymerases, time-resolved fluorescence spectroscopy was used to examine the interaction between the Klenow fragment of DNA polymerase I and synthetic DNA primer-templates containing extrahelical bases at defined positions within the template strand. A dansyl probe attached to the DNA was used to measure the fractional occupancies of the polymerase and 3'-5' exonuclease sites of the enzyme for DNA substrates with and without the extrahelical bases. The presence of an extrahelical base at the first position from the primer 3' terminus increased the level of partitioning of the DNA substrates into the 3'-5' exonuclease site by 3-7-fold, relative to the perfectly base-paired primer-template, depending on the identity of the extrahelical base. The ability of different extrahelical bases to promote partitioning of DNA into the 3'-5' exonuclease site decreased in the following order: G > A approximately T > C. The results of partitioning measurements for DNA substrates containing a bulged adenine base at different positions within the template showed that an extrahelical base is recognized up to five bases from the primer 3' terminus. The largest effects were observed for the extrahelical base at the third or fourth positions from the primer terminus, which increased the level of partitioning of DNA into the 3'-5' exonuclease site by 8- and 18-fold, respectively, relative to that of the perfectly base-paired substrate. Steady-state fluorescence measurements of analogous primer-templates containing 2-aminopurine (AP) at the primer 3' terminus indicate that extrahelical bases increase the degree of terminus unwinding, especially when close to the terminus. In addition, steady-state kinetic measurements of removal of AP from the primer-templates indicate that the exonucleolytic cleavage activity of Klenow fragment is correlated with the increased level of partitioning of bulged DNA substrates to the 3'-5' exonuclease site relative to that of properly base-paired DNA. The results of this study indicate that misalignment of primer and template strands to generate an extrahelical base strongly promotes transfer of a DNA substrate to the 3'-5' exonuclease site, suggesting that the premutational intermediates in frameshift mutagenesis are subject to proofreading by the polymerase.     

Using the fluorescence decay of 2-aminopurine to investigate conformational change in the recognition sequence of the EcoRV DNA-(adenine-N6)-methyltransferase on enzyme binding

The EcoRV DNA methyltransferase methylates the first adenine in the GATATC recognition sequence. It is presumed that methylation proceeds by a nucleotide flipping mechanism but no crystal structure is available to confirm this. A popular solution-phase assay for nucleotide flipping employs the fluorescent adenine analogue, 2-aminopurine (2AP), substituted at the methylation target site; a substantial increase in fluorescence intensity on enzyme binding indicates flipping. However, this appeared to fail for M.EcoRV, since 2AP substituted for the non-target adenine in the recognition sequence showed a much greater intensity increase than 2AP at the target site. This anomaly is resolved by recording the fluorescence decay of 2AP which shows that the target 2AP is indeed flipped by the enzyme, but its fluorescence is quenched by interaction with aromatic residues in the catalytic site, whereas bending of the duplex at the non-target site alleviates inter-base quenching and exposes the 2AP to solvent.     

Interactions of pyronin Y(G) with nucleic acids

Spectral properties of pyronin Y(PY) alone or in complexes with natural and synthetic nucleic acids of various base compositions have been studied in aqueous solution containing 10 or 150 mM NaCl and 5 mM Hepes at pH 7.0. The dimerization constant (KD = 6.27 X 10(3), M-1) and the absorption spectra of the dye in monomeric and dimeric form were established. The complexes of PY with single-stranded (ss) nucleic acids show a hypsochromic shift in absorption, and their fluorescence is quenched by over 90% compared to free dye. In contrast, complexes with double-stranded (ds) RNA or DNA (binding by intercalation) exhibit a bathochromic shift in their absorption (excitation) spectrum, and their fluorescence is correlated with the base composition of the binding site. Namely, guanine quenches fluorescence of PY by up to 90%, whereas A, C, I, T, and U bases exert a rather minor effect on the fluorescence quantum yield of the dye. The intrinsic association constant of the dye to ds RNA (Ki = 6.96 X 10(4), M-1) and to ds DNA (Ki = 1.74 X 10(4), M-1) was measured in 150 mM NaCl; the binding site size was 2-3 base pair for both polymers. Implications of these findings for qualitative and quantitative cytochemistry of nucleic acids are discussed.     

DNA substrate structural requirements for the exonuclease and polymerase activities of procaryotic and phage DNA polymerases

A DNA duplex covalently cross-linked between specific bases has been prepared. This and similar duplexes are substrates for the polymerase and exonuclease activities of the Klenow fragment of Escherichia coli DNA polymerase I and T4 and T7 DNA polymerases. The action of Klenow fragment on these duplexes indicates that the polymerase site does not require that the DNA duplex undergo strand separation for activity, whereas the exonuclease site requires that at least four base pairs of the primer strand must melt out for the exonucleolytic removal of nucleotides from the primer terminus. The exonucleolytic action of T4 and T7 DNA polymerases requires that only two and three bases respectively melt out for excision of nucleotides from the primer terminus. Klenow fragment and T4 DNA polymerase are able to polymerize onto duplexes incapable of strand separation, whereas T7 DNA polymerase seems to require that the primer terminus be at least three bases from the cross-linked base pair. A DNA duplex with a biotin covalently linked to a specific base has been prepared. In the presence of the biotin binding protein avidin, the exonucleolytic activity of Klenow fragment requires that the primer terminus be at least 15 base pairs downstream from the base with the biotin-avidin complex. On the other hand, the polymerase activity of Klenow fragment required that the primer terminus be at least six base pairs downstream from the base with the biotin-avidin complex. These results suggest that the polymerase and exonuclease sites of Klenow are physically separate in solution and exhibit different substrate structural requirements for activity.     

AP site structural determinants for Fpg specific recognition.

The binding of Escherichia coli and Lactococcus lactis Fapy-DNA glyosylase (Fpg) proteins to DNA containing either cyclic or non-cyclic abasic (AP) site analogs was investigated by electrophoretic mobility shift assay (EMSA) and by footprinting experiments. We showed that the reduced AP site is the best substrate analog for the E.coli and L.lactis enzymes ( K Dapp = 0.26 and 0.5 nM, respectively) as compared with the other analogs tested in this study ( K Dapp >2.8 nM). The 1,3-propanediol (Pr) residue-containing DNA seems to be the minimal AP site structure allowing a Fpg specific DNA binding, since the ethyleneglycol residue is not specifically bound by these enzymes. The newly described cyclopentanol residue is better recognized than tetrahydrofuran (for the E.coli Fpg, K Dapp = 2.9 and 25 nM, respectively). These results suggest that the hemiacetal form of the AP site is negatively discriminated by the Fpg protein suggesting a hydrogen bond between the C4'-hydroxyl group of the sugar and a Fpg residue. High-resolution hydroxyl radical footprinting using a duplex containing Pr shows that Fpg binds to six nucleotides on the strand containing the AP site and only the base opposite the lesion on the undamaged complementary strand. This comparative study provides new information about the molecular mechanism involved in the Fpg AP lyase activity.     

Probing DNA polymerase-DNA interactions: examining the template strand in exonuclease complexes using 2-aminopurine fluorescence and acrylamide quenching

The bacteriophage T4 DNA polymerase forms fluorescent complexes with DNA substrates labeled with 2-aminopurine (2AP) in the template strand; the fluorescence intensity depends on the position of 2AP. When preexonuclease complexes are formed, complexes at the crossroads between polymerase and exonuclease complexes, 2AP in the +1 position in the template strand is fully free of contacts with the adjacent bases as indicated by high fluorescence intensity and a long fluorescence lifetime of about 10.9 ns. Fluorescence intensity decreases for 2AP in the template strand when the primer end is transferred to the exonuclease active center to form exonuclease complexes, which indicates a change in DNA conformation; 2AP can now interact with adjacent bases, which quenches fluorescence emission. Some polymerase-induced base unstacking for 2AP in the template strand in exonuclease complexes is observed but is restricted primarily to the n and +1 positions, which indicates that the DNA polymerase holds the template strand in a way that forces base unstacking only in a small region near the primer terminus. A hold on the template strand will help to maintain the correct alignment of the template and primer strands during proofreading. Acrylamide quenches 2AP fluorescence in preexonuclease and in exonuclease complexes formed with DNA labeled with 2AP in the template strand, which indicates that the template strand remains accessible to solvent in both complexes. These studies provide new information about the conformation of the template strand in exonuclease complexes that is not available from structural studies.     

2-Aminopurine fluorescence studies of base stacking interactions at abasic sites in DNA: metal-ion and base sequence effects.

Metal-ion and sequence dependent changes in the stacking interactions of bases surrounding abasic (AB) sites in 10 different DNA duplexes were examined by incorporating the fluorescent nucleotide probe 2-aminopurine (2-AP), opposite to the site (AB-APopp) or adjacent to the site (AB-APadj) on either strand. A detailed study of the fluorescence emission and excitation spectra of these AB duplexes and their corresponding parent duplexes indicates that AB-APoppis significantly less stacked than 2-AP in the corresponding normal duplex. In general, AB-APadjon the AB strand is stacked, but AB-APadjon the opposite strand shows destabilized stacking interactions. The results also indicate that divalent cation binding to the AB duplexes contributes to destabilizaton of the base stacking interactions of AB-APopp, but has little or no effect on the stacking interactions of AB-APadj. Consistent with these results, the fluorescence of AB-APoppis 18-30-fold more sensitive to an externally added quenching agent than the parent normal duplex. When uracil DNA glycosylase binds to AB-APoppin the presence of 2.5 mM MgCl2, a 3-fold decrease in fluorescence is observed ( K d = 400 +/- 90 nM) indicating that the unstacked 2-APoppbecomes more stacked upon binding. On the basis of these fluorescence studies a model for the local base stacking interactions at these AB sites is proposed.