Distinguishing "looped-out" and "stacked-in" DNA bulge conformation using fluorescent 2-aminopurine replacing a purine base

The conformation of a bulged DNA base, whether looped-out of the DNA helix or stacked-in between the flanking bases, can be distinguished using fluorescence spectroscopy of an inserted fluorescent base. If 2-aminopurine, a structural analog of adenine and guanine, is placed in duplex DNA as the bulged base replacing an adenine or guanine, it loops out of the DNA helix into solution. This is determined by the decrease or increase of 2-aminopurine fluorescence during DNA thermomelting: if the 2-aminopurine base stacks into the helix, its fluorescence increases or remains about the same during DNA duplex melting, but if the 2-aminopurine base loops out of the helix, its fluorescence decreases upon melting of the DNA duplex.     

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.     

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.     

Stacking-unstacking dynamics of oligodeoxynucleotide trimers

The structure and dynamics of DNA trimers are experimentally assessed using the fluorescent purine analogue 2-aminopurine (2AP), incorporating 2AP between purine and pyrimidine bases to form 5'dXp2APpY3' molecules. Circular dichroism and fluorescence quenching of the 2AP show that the bases are stacked; at the same time, fluorescence decay lifetimes are heterogeneous, indicative of conformational sampling. 2AP does not exhibit the long fluorescence decay time characteristic of the free nucleoside, suggesting that its motions in the trimers bring it into proximity of the neighboring bases, resulting in efficient charge transfer and average fluorescence lifetimes on the order of 1-2 ns.     

2-aminopurine fluorescence: discrimination between specific and unspecific ligand binding to the kissing-loop dimer of the HIV-1 RNA

The fluorescent 2-aminopurine probe (2-AP) incorporated into the loop of 23-mer RNA hairpin of HIV-1 genome dimerization initiation site (DIS) was used for discrimination of specific and unspecific binding of paromomycin and spermine to the kissing loop dimer (KD) formed in solution. While both ligands stabilized the KD RNA structure, only paromomycin binding resulted in significant increase of 2-AP fluorescence. These observations suggest that the 2-AP fluorescent RNA construct might be useful for selecting ligands specifically binding the HIV-1 kissing loop RNA dimer.     

Conformational changes that occur during an RNA-editing adenosine deamination reaction

ADARs are adenosine deaminases responsible for RNA-editing reactions that occur within duplex RNA. Currently little is known regarding the nature of the protein-RNA interactions that lead to site-selective adenosine deamination. We previously reported that ADAR2 induced changes in 2-aminopurine fluorescence of a modified substrate, consistent with a base-flipping mechanism. Additional data have been obtained using full-length ADAR2 and a protein comprising only the RNA binding domain (RBD) of ADAR2. The increase in 2-aminopurine fluorescence is specific to the editing site and dependent on the presence of the catalytic domain. Hydroxyl radical footprinting demonstrates that the RBD protects a region of the RNA duplex around the editing site, suggesting a significant role for the RBD in identifying potential ADAR2 editing sites. Nucleotides near the editing site on the non-edited strand become hypersensitive to hydrolytic cleavage upon binding of ADAR2 RBD. Therefore, the RBD may assist base flipping by increasing the conformational flexibility of nucleotides in the duplex adjacent to its binding site. In addition, an increase in tryptophan fluorescence is observed when ADAR2 binds duplex RNA, suggesting a conformational change in the catalytic domain of the enzyme. Furthermore, acrylamide quenching experiments indicate that RNA binding creates heterogeneity in the solvent accessibility of ADAR2 tryptophan residues, with one out of five tryptophans more solvent-accessible in the ADAR2.RNA complex.     

A base change in the catalytic core of the hairpin ribozyme perturbs function but not domain docking

The hairpin ribozyme is a small endonucleolytic RNA motif with potential for targeted RNA inactivation. It optimally cleaves substrates containing the sequence 5'-GU-3' immediately 5' of G. Previously, we have shown that tertiary structure docking of its two domains is an essential step in the reaction pathway of the hairpin ribozyme. Here we show, combining biochemical and fluorescence structure and function probing techniques, that any mutation of the substrate base U leads to a docked RNA fold, yet decreases cleavage activity. The docked mutant complex shares with the wild-type complex a common interdomain distance as measured by time-resolved fluorescence resonance energy transfer (FRET) as well as the same solvent-inaccessible core as detected by hydroxyl-radical protection; hence, the mutant complex appears nativelike. FRET experiments also indicate that mutant docking is kinetically more complex, yet with an equilibrium shifted toward the docked conformation. Using 2-aminopurine as a site-specific fluorescent probe in place of the wild-type U, a local structural rearrangement in the substrate is observed. This substrate straining accompanies global domain docking and involves unstacking of the base and restriction of its conformational dynamics, as detected by time-resolved 2-aminopurine fluorescence spectroscopy. These data appear to invoke a mechanism of functional interference by a single base mutation, in which the ribozyme-substrate complex becomes trapped in a nativelike fold preceding the chemical transition state.     

2-Aminopurine Fluorescence: Discrimination Between Specific and Unspecific Ligand Binding to the Kissing-Loop Dimer of the HIV-1 RNA

Abstract

The fluorescent 2-aminopurine probe (2-AP) incorporated into the loop of 23-mer RNA hairpin of HIV-1 genome dimerization initiation site (DIS) was used for discrimination of specific and unspecific binding of paromomycin and spermine to the kissing loop dimer (KD) formed in solution. While both ligands stabilized the KD RNA structure, only paromomycin binding resulted in significant increase of 2-AP fluorescence. These observations suggest that the 2-AP fluorescent RNA construct might be useful for selecting ligands specifically binding the HIV-1 kissing loop RNA dimer.     

Effect of aminoglycoside antibiotics on the conformation of the unpaired loop adenine of avian leucosis virus RNA as revealed using 2-aminopurine fluorescence

The fluorescent properties of 2-aminopurine (2-AP) incorporated in an RNA sequence are used to study the structural dynamics and local changes of the retroviral RNA structure. Using 2-AP, the conformational states of the unpaired loop adenine in avian leucosis virus RNA were studied upon its interaction with aminoglycoside antibiotics. The intensity of 2-AP fluorescence in the monomeric RNA hairpin was higher than in both RNA dimers. The intensity of fluorescence in the extended dimer was significantly lower than in the kissing loop dimer. The finding was be explained by the fact that stacking contacts in the extended dimer produce a more compact loop structure than in the kissing loop dimer. When the binding of aminogycoside antibiotics with the kissing loop dimer RNA was analyzed, only tobramycin increased the intensity of 2-AP fluorescence almost threefold. The results showed that 2-AP fluorescence is suitable for detecting local changes in complexes of retroviral RNA with ligands.     

2-Aminopurine as a real-time probe of enzymatic cleavage and inhibition of hammerhead ribozymes.

The design, synthesis and study of internally fluorescent hammerhead (HH) ribozymes, where changes in fluorescence parameters directly reflect the progress of the ribozyme's cleavage chemistry, are described. The approach relies on a HH substrate modified at position 1.1, proximal to the cleavage site, with 2-aminopurine (2AP), an intensely fluorescent adenosine isoster. The incorporation of 2AP, an unnatural nucleoside, does not interfere with the ribozyme folding and catalysis. Since 2AP is highly sensitive to environmental changes, its fluorescence is dramatically altered upon ribozyme-mediated cleavage of the substrate. This generates a measurable signal that directly reflects the progress of the ribozyme's reaction in real time. Identical pseudo first order rate constants are obtained for HH constructs using both continuous fluorescence monitoring and radioactive labeling. This rapid and real-time monitoring facilitates the study of ribozyme activity under different conditions (e.g., ionic strength, pH, etc.), and provides a useful assay to rapidly screen potential inhibitors. Three hitherto unknown HH inhibitors are presented and compared to neomycin B and chlortetracycline, two previously studied HH inhibitors. All three new small molecules, neo-acridine, guanidino-neomycin B, and [Delta-(Eilatin)Ru(bpy)(2)](2+), prove to be better inhibitors than neomycin B or chlortetracycline. Investigating HH inhibition under different ionic strengths reveals that the binding of neo-acridine, [Delta-(Eilatin)Ru(bpy)(2)](2+), and chlortetracycline to the HH involves hydrophobic interactions as their RNA affinities are largely unaffected by increasing salt concentrations. In contrast, neomycin B loses more than 50-fold of its inhibitory ability as the NaCl concentration is increased from 50 to 500mM.     

Influence of base stacking and hydrogen bonding on the fluorescence of 2-aminopurine and pyrrolocytosine in nucleic acids

Fluorescent nucleobase analogues are used extensively to probe the structure and dynamics of nucleic acids. The fluorescence of the adenine analogue 2-aminopurine and the cytosine analogue pyrrolocytosine is significantly quenched when the bases are located in regions of double-stranded nucleic acids. To allow more detailed structural information to be obtained from fluorescence studies using these bases, we have studied the excited-state properties of the bases at the CIS and TDB3LYP level in hydrogen-bonded and base-stacked complexes. The results reveal that the first excited state (the fluorescent state) of a hydrogen-bonded complex containing 2-aminopurine and thymine is just the first excited state of 2-aminopurine alone. However, the same cannot be said for structures in which 2-aminopurine is base stacked with other nucleobases. Stacking causes the molecular orbitals involved in the fluorescence transition to spread over more than one base. The predicted rate for the fluorescence transition is reduced, thus reducing the fluorescence quantum yield. The decrease in radiative rate varies with the stacking arrangement (e.g., A- or B-form DNA) and with the identity of the nucleobase with which 2-aminopurine is stacked. Stacking 2-aminopurine between two guanine moieties is shown to significantly decrease the energy gap between the first and second excited states. We do not find reliable evidence for a low-energy charge-transfer state in any of the systems that were studied. In the case of pyrrolocytosine, base stacking was found to reduce the oscillator strength for the fluorescence transition, but very little spreading of molecular orbitals across more than one base was observed.     

Hybridization of 2'-O-methyl and 2'-deoxy molecular beacons to RNA and DNA targets

Molecular beacons are stem-loop hairpin oligonucleotide probes labeled with a fluorescent dye at one end and a fluorescence quencher at the other end; they can differentiate between bound and unbound probes in homogeneous hybridization assays with a high signal-to-background ratio and enhanced specificity compared with linear oligonucleotide probes. However, in performing cellular imaging and quantification of gene expression, degradation of unmodified molecular beacons by endogenous nucleases can significantly limit the detection sensitivity, and results in fluorescence signals unrelated to probe/target hybridization. To substantially reduce nuclease degradation of molecular beacons, it is possible to protect the probe by substituting 2'-O-methyl RNA for DNA. Here we report the analysis of the thermodynamic and kinetic properties of 2'-O-methyl and 2'-deoxy molecular beacons in the presence of RNA and DNA targets. We found that in terms of molecular beacon/target duplex stability, 2'-O-methyl/RNA > 2'-deoxy/RNA > 2'-deoxy/DNA > 2'-O-methyl/DNA. The improved stability of the 2'-O-methyl/RNA duplex was accompanied by a slightly reduced specificity compared with the duplex of 2'-deoxy molecular beacons and RNA targets. However, the 2'-O-methyl molecular beacons hybridized to RNA more quickly than 2'-deoxy molecular beacons. For the pairs tested, the 2'-deoxy-beacon/DNA-target duplex showed the fastest hybridization kinetics. These findings have significant implications for the design and application of molecular beacons.     

Intrinsic flexibility of snRNA hairpin loops facilitates protein binding

Stem–loop II of U1 snRNA and Stem–loop IV of U2 snRNA typically have 10 or 11 nucleotides in their loops. The fluorescent nucleobase 2-aminopurine was used as a substitute for the adenines in each loop to probe the local and global structures and dynamics of these unusually long loops. Using steady-state and time-resolved fluorescence, we find that, while the bases in the loops are stacked, they are able to undergo significant local motion on the picosecond/nanosecond timescale. In addition, the loops have a global conformational change at low temperatures that occurs on the microsecond timescale, as determined using laser T-jump experiments. Nucleobase and loop motions are present at temperatures far below the melting temperature of the hairpin stem, which may facilitate the conformational change required for specific protein binding to these RNA loops.     

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.     

Lethal mutagenesis of poliovirus mediated by a mutagenic pyrimidine analogue

Lethal mutagenesis is the mechanism of action of ribavirin against poliovirus (PV) and numerous other RNA viruses. However, there is still considerable debate regarding the mechanism of action of ribavirin against a variety of RNA viruses. Here we show by using T7 RNA polymerase-mediated production of PV genomic RNA, PV polymerase-catalyzed primer extension, and cell-free PV synthesis that a pyrimidine ribonucleoside triphosphate analogue (rPTP) with ambiguous base-pairing capacity is an efficient mutagen of the PV genome. The in vitro incorporation properties of rPTP are superior to ribavirin triphosphate. We observed a log-linear relationship between virus titer reduction and the number of rPMP molecules incorporated. A PV genome encoding a high-fidelity polymerase was more sensitive to rPMP incorporation, consistent with diminished mutational robustness of high-fidelity PV. The nucleoside (rP) did not exhibit antiviral activity in cell culture, owing to the inability of rP to be converted to rPMP by cellular nucleotide kinases. rP was also a poor substrate for herpes simplex virus thymidine kinase. The block to nucleoside phosphorylation could be bypassed by treatment with the P nucleobase, which exhibited both antiviral activity and mutagenesis, presumably a reflection of rP nucleotide formation by a nucleotide salvage pathway. These studies provide additional support for lethal mutagenesis as an antiviral strategy, suggest that rPMP prodrugs may be highly efficacious antiviral agents, and provide a new tool to determine the sensitivity of RNA virus genomes to mutagenesis as well as interrogation of the impact of mutational load on the population dynamics of these viruses.     

Structure and dynamics of a fluorescent DNA oligomer containing the EcoRI recognition sequence: fluorescence, molecular dynamics, and NMR studies

The self-complementary DNA decamer duplex d(CTGAATTCAG)2 and its modified counterpart d(CTGA[2AP]TTCAG)2, where the innermost adenine (6-aminopurine) has been replaced with the fluorescent analogue 2-aminopurine (2AP), have been studied by fluorescence and NMR spectroscopy and simulated by molecular dynamics. Both decamers are recognized and cleaved by the EcoRI restriction endonuclease. 2D NMR results show that both decamers have a standard B-type conformation below 20 degrees C, though a disturbance exists to the 5' side of the 2AP site which may originate from increased local mobility. The fluorescence and fluorescence anisotropy decays of both decamers, as well as the one containing 2AP in only one chain, were studied as a function of temperature. The data show that the 2AP base exists in a temperature-dependent distribution of states and shows rapid motions, suggesting interconversion among these states on a time scale of about 10(-10) s. The integrated fluorescence of the decamer with 2AP in both chains shows a large increase around the helix melting temperature whereas the decamer with one 2AP shows only a mild increase, showing that the mixed helix has a different structural transition as sensed by the 2AP base. The data suggest a model of conformational states which have distinct fluorescence decay times. The various states may differ in the degree of base stacking. Fluctuations in the degree of stacking of the A or 2AP base are supported by molecular dynamics simulations, which additionally show that the 2AP-T or A-T base pair hydrogen bonds remain intact during these large motions.     

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.     

2,6-Diaminopurine nucleoside derivative of 9-ethyloxy-2-oxo-1,3-diazaphenoxazine (2-amino-Adap) for recognition of 8-oxo-dG in DNA

8-Oxo-2′-deoxyguanosine (8-oxo-dG) is a nucleoside resulting from oxidative damage and is known to be mutagenic. 8-Oxo-dG has been related to aging and diseases, including neurological disorders and cancer. Recently, we reported that a fluorescent nucleoside derivative, adenosine-1,3-diazaphenoxazine (Adap), forms a stable base pair with 8-oxo-dG in DNA with accompanying efficient quenching. In this study, a new Adap derivative having an additional 2-amino group on the adenosine moiety (2-amino-Adap) was designed with the anticipation of additional hydrogen bonding with the 8-oxo group of 8-oxo-dG. The properties of the ODN containing 2-amino-Adap were evaluated by measuring thermal stability and fluorescence quenching. In contrast to the previously designed Adap, the base-pairing and fluorescence quenching properties of 2-amino-Adap varied depending on the ODN sequence, and there was no clear indication of an additional hydrogen bond with 8-oxo-dG. Instead, the base pairing of 2-amino-Adap with dG was significantly destabilized compared with that of Adap with dG, resulting in improved selectivity for 8-oxo-dG in the human telomere DNA sequence. Thus, the telomere-targeting ODN probe containing 2-amino-Adap displayed selective, sensitive and quantitative detection of 8-oxo-dG in the human telomere DNA sequence in a light-up detection system using SYBR Green.