7-deazaadenine and 7-deazaguanine

RNA structures are very dynamic and the dynamic motions result in a heterogeneous conformational ensemble. It is crucial to illustrate the role of conformational dynamics in RNA function. A variety of spectroscopic methods have been used to investigate the dynamic aspects of RNA structures. Recently, ultrafast time-resolved spectroscopy, a well-established technique, has been introduced as a new tool in this field. With femtosecond time-resolution, one can resolve the heterogeneous nature of RNA conformational ensemble quantitatively, detect and characterize minor unusual conformations, and capture folding events that may occur on a fast timescale. Here, we review the technical aspects of using an ultrafast fluorescence up-conversion technique to probe the heterogeneous base stacking patterns in RNA motifs and dynamic base motions that allow sampling of multiple states.

Topics: 2-Aminopurine; Adenine; Anisotropy; Equipment Design; Guanine; Models, Molecular; Models, Statistical; Molecular Conformation; Molecular Structure; Nucleic Acid Conformation; RNA; RNA, Viral; Spectrometry, Fluorescence; Time Factors

Nucleic acids studies use 7-deazaguanine (G*) and 7-deazaadenine (A*) as analogues of natural purine bases incapable of forming Hoogsteen base pairs, which prevents them from being involved in DNA triplexes and tetraplexes. Reduced propensity of the G*- and/or A*-modified DNA to form alternative DNA structures is utilized, for example, in PCR amplification of guanine-rich sequences. Both G* and A* exhibit significantly lower potentials of their oxidation, compared to the respective natural nucleobases. At carbon electrodes, A* yields an oxidation peak which is by about 200-250 mV less positive than the peak due to adenine, but coincides with oxidation peak produced by natural guanine residues. On the other hand, oxidation signal of G* occurs at a potential by about 300 mV less positive than the peak due to guanine, being well separated from electrochemical signals of any natural DNA component. We show that enzymatic incorporation of G* and A* can easily be monitored by simple ex situ voltammetric analysis of the modified DNA at carbon electrodes. Particularly G* is shown as an attractive electroactive marker for DNA, efficiently incorporable by PCR. While densely G*-modified DNA fragments exhibit strong quenching of fluorescence of SYBR dyes, commonly used as fluorescent indicators in both gel staining and real time PCR applications, the electrochemical detection provides G*-specific signal suitable for the quantitation of the amplified DNA as well as for the determination of the DNA modification extent. Determination of DNA amplicons based on the measurement of peak G*(ox) is not affected by signals produced by residual oligonucleotide primers or primary templates containing natural purines.

Topics: Adenine; Carbon; DNA; Electrochemical Techniques; Electrodes; Enzymes; Guanine; Oxidation-Reduction; Polymerase Chain Reaction

DNA damage that stalls replicative polymerases can be bypassed with the Y-family polymerases. These polymerases have more open active sites that can accommodate modified nucleotides. The lack of protein-DNA interactions that select for Watson-Crick base pairs correlate with the lowered fidelity of replication. Interstrand hydrogen bonds appear to play a larger role in dNTP selectivity. The mechanism by which purine-purine mispairs are formed and extended was examined with Solfolobus solfataricus DNA polymerase IV, a member of the RAD30A subfamily of the Y-family polymerases, as is pol eta. The structures of the purine-purine mispairs were examined by comparing the kinetics of mispair formation with adenine versus 1-deaza- and 7-deazaadenine and guanine versus 7-deazaguanine at four positions in the DNA, the incoming dNTP, the template base, and both positions of the terminal base pair. The time course of insertion of a single dNTP was examined with a polymerase concentration of 50 nM and a DNA concentration of 25 nM with various concentrations of dNTP. The time courses were fitted to a first-order equation, and the first-order rate constants were plotted against the dNTP concentration to produce k pol and K d (dNTP) values. A decrease in k pol/ K d (dNTP) associated with the deazapurine substitution would indicate that the position is involved in a crucial hydrogen bond. During correct base pair formation, the adenine to 1-deazaadenine substitution in both the incoming dNTP and template base resulted in a >1000-fold decrease in k pol/ K d (dNTP), indicating that interstrand hydrogen bonds are important in correcting base pair formation. During formation of purine-purine mispairs, the k pol/ K d (dNTP) values for the insertion of dATP and dGTP opposite 7-deazaadenine and 7-deazaguanine were decreased >10-fold with respect to those of the unmodified nucleotides. In addition, the rate of incorporation of 1-deaza-dATP opposite guanine was decreased 5-fold. These results suggest that during mispair formation the newly forming base pair is in a Hoogsteen geometry with the incoming dNTP in the anti conformation and the template base in the syn conformation. These results indicate that Dpo4 holds the incoming dNTP in the normal anti conformation while allowing the template nucleotide to change conformations to allow reaction to occur. This result may be functionally relevant in the replication of damaged DNA in that the polymerase may allow the template to adopt

Topics: Adenine; Archaeal Proteins; Base Pair Mismatch; Base Pairing; DNA Damage; DNA Polymerase beta; DNA Replication; Guanine; Hydrogen Bonding; Kinetics; Molecular Structure; Purines; Sulfolobus solfataricus

The mechanism by which purine-purine mispairs are formed and extended was examined with the high-fidelity Klenow fragment of Escherichia coli DNA polymerase I with the proofreading exonuclease activity inactivated. The structures of the purine-purine mispairs were examined by comparing the kinetics of mispair formation with adenine versus 7-deazaadenine and guanine versus 7-deazaguanine at four positions in the DNA, the incoming dNTP, the template base, and both positions of the terminal base pair. A decrease in rate associated with a 7-deazapurine substitution would suggest that the nucleotide is in a syn conformation in a Hoogsteen base pair with the opposite base. During mispair formation, the k(pol)/K(d) values for the insertion of dATP opposite A (dATP/A) as well as dATP/G and dGTP/G were decreased greater than 10-fold with the deazapurine in the dNTP. These results suggest that during mispair formation the newly forming base pair is in a Hoogsteen geometry with the incoming dNTP in the syn conformation and the template base in the anti conformation. During mispair extension, the only decrease in k(pol)/K(d) was associated with the G/G base pair in which 7-deazaguanine was in the template strand. These results as well as previous results [McCain et al. (2005) Biochemistry 44, 5647-5659] in which a hydrogen bond was found between the 3-position of guanine at the primer terminus and Arg668 during G/A and G/G mispair extension indicate that the conformation of the purine at the primer terminus is in the anti conformation during mispair extension. These results suggest that purine-purine mispairs are formed via a Hoogsteen geometry in which the dNTP is in the syn conformation and the template is in the anti conformation. During extension, however, the conformation of the primer terminus changes to an anti configuration while the template base may be in either the syn or anti conformations.

Topics: Adenine; Base Pair Mismatch; Base Sequence; DNA Polymerase I; DNA Replication; Escherichia coli; Guanine; Hydrogen Bonding; Kinetics; Molecular Sequence Data; Nucleic Acid Conformation; Purines; Structure-Activity Relationship

The 7-deaza analogues of guanine and adenine were incorporated into polymerase chain reaction (PCR) products by substitution of the appropriate nucleotide triphosphates into the reaction. These PCR products can be immobilized on ITO electrodes and detected by catalytic cyclic voltammetry with ruthenium polypyridyl complexes. Immobilization on indium tin oxide (ITO) electrodes of 330- and 1200-base pair (bp) PCR amplicons from the E. coli dacA gene containing one or both of the 7-deazapurines was effected by precipitation from a 9:1 DMF/acetate solution. Amplicons containing the 7-deazaguanine base were detected by observing current enhancement in the cyclic voltammogram of Ru(dmb)3(3)+/2+ (dmb = 4,4'-dimethyl-2,2'-bipyridine) due to the selective oxidation of the modified base by this mediator. Oxidation of incorporated 7-deazaadenine bases in addition to native guanines gives rise to a higher current enhancement in the cyclic voltammogram of Ru(bpy)3(3)+/2+ (bpy = 2,2'-bipyridine) compared to the enhancement observed in the presence of guanine only. This strategy was employed to simultaneously detect the 330-bp sequence containing 7-deazaadenine and the 1200-bp sequence containing 7-deazaguanine on the same ITO electrode. Such a strategy may provide a means for detecting multiple genes on a single microlocation and may thereby lead to more highly multiplexed gene assays.

Topics: Adenine; Bacterial Proteins; Carrier Proteins; DNA; Electrochemistry; Electrodes; Escherichia coli; Guanine; Hexosyltransferases; Muramoylpentapeptide Carboxypeptidase; Oxidation-Reduction; Penicillin-Binding Proteins; Peptidyl Transferases; Polymerase Chain Reaction

HMG-domain proteins bind strongly to bent DNA structures, including cruciform and cisplatin-modified duplexes. Such protein-platinated DNA complexes, formed where the DNA is modified by the active cis but not the inactive trans isomer of diamminedichloroplatinum(II), are implicated in the cytotoxic mechanism of the drug. A series of oligonucleotide duplexes with deoxyguanosine nucleosides flanking a cis-[Pt(NH(3))(2)¿d(GpG)-N7(1),-N7(2)¿] cross-link have been synthesized. These probes were used to determine the flanking sequence dependence of the affinity of the individual HMG domains of HMG1 toward cisplatin-modified DNA. Nine related sequences, where N(1) and N(2) are not dG and GG is the 1,2-intrastrand cisplatin adduct in N(1)GGN(2), were previously investigated [Dunham, S. U., and Lippard, S. J. (1997) Biochemistry 36, 11428-11436]. Three of the seven remaining possible sequences for which N(1) and/or N(2) was dG were prepared here by using normal deoxyguanosine, but the rest, where N(1) is dG and N(2) is dA, dC, T, or dG, could not be isolated in pure form. These sequences were accessed by using the synthetic bases 7-deazaadenine and 7-deazaguanine, which lack the nucleophilic N7 atom in the purine ring. Deaza nucleotides accurately mimic the properties of the natural bases, allowing the interaction of the HMG-domain proteins with cisplatin-modified DNA to be examined. These experiments reveal that the flexibility of A.T versus G.C flanking base pairs, rather than base-specific contacts, determines HMG1domA protein selectivity. This conclusion was supported by use of mutant HMG1domA and HMG1domB proteins, which exhibit identical flanking sequence selectivity. The methods and results obtained here not only improve our understanding of how proteins might mediate cisplatin genotoxicity but also should apply more generally in the investigation of how other proteins interact with damaged DNA.

Topics: Adenine; Base Sequence; Cisplatin; Deoxyguanosine; Dinucleoside Phosphates; DNA Adducts; DNA Probes; Guanine; High Mobility Group Proteins; Mutagenesis, Site-Directed; Nucleic Acid Heteroduplexes; Oligonucleotides; Protein Binding; Protein Structure, Tertiary; Recombinant Proteins

Telomeres are required for eukaryotic chromosome stability. They consist of regularly repeating guanine-rich sequences, with a single-stranded 3' terminus. Such sequences have been demonstrated to have the propensity to adopt four-stranded structures based on a tetrad of guanine bases. The formation of an intramolecular foldback tetraplex is associated with markedly increased mobility in polyacrylamide. Most telomeric sequences are based either on a repeat of d(TnGGGG) or d(TnAGGG) sequences. We have used a combination 7-deazaguanine or 7-deaza-adenine substitution, chemical modification and gel electrophoresis to address the following aspects of intramolecular tetraplex formation. (i) Intramolecular tetraplex formation by d(TTTTGGGG)4 sequences is prevented by very low levels of 7-deazaguanine substitution. This confirms the important role of guanine N7 in the formation of the tetraplex. (ii) The sequences d(TTAGGG)4 and d(TTTTAGGG)4 fold into tetraplexes. By contrast, the electrophoretic behaviour of d(TTTTGGGA)4, d(TTTTAGAG)4 and d(TTTTGAGA)4 does not indicate formation of stable intramolecular tetraplexes under available conditions. (iii) Selective 7-deazaguanine and 7-deaza-adenine substitutions in d(TTTTAGGG)4 give results consistent with tetraplex folding by the formation of three G4 tetrads, with the adenine bases formally part of the single-stranded loops, where they probably interact with thymine bases. These results demonstrate that eukaryotic cells appear to have selected just those sequences that can adopt the tetraplex conformation for their telomeres, while those that cannot have been avoided. This suggests that the conformation may be significant in the function of the telomere, such as attachment to nuclear structures.

Topics: Adenine; Base Sequence; DNA; Guanine; Molecular Sequence Data; Nucleic Acid Conformation; Telomere

To study the mechanism of arrest of DNA synthesis at d(TC)n and d(GA)n sequences, single-stranded DNA molecules including d(TC)27 or d(TC)31 tracts or a d(GA)27 tract were used as templates for in vitro assays of complementary DNA synthesis performed by extension of a primer with the Klenow polymerase or the Taq polymerase (Thermus aquaticus DNA polymerase). Electrophoresis of the products revealed that arrests occurred around the middle of these tracts. The arrests in the d(TC)n sequences were eliminated when dATP or dGTP was replaced with the analogue 7-deaza dATP or 7-deaza dGTP, respectively, or when the templates were preincubated with the Escherichia coli single-strand binding protein (SSB). Preincubation of the template including a d(GA)27 tract with SSB has also eliminated the arrests at this sequence. Furthermore, arrests did not occur at d[G(7-deaza A)]27 or d[(7-deaza G)A]27 tracts when molecules including such tracts were used as templates. These results are compatible with the notion that the arrests were caused by formation of d(TC)i.d(GA)i.d(TC)i and d(GA)i.d(GA)i.d(TC)i triplexes, in which the bases in the uncopied portions of the d(TC)n tracts, or of the d(GA)27 tract, and the purine bases in the newly synthesized d(TC)i.d(GA)i duplexes were bound by hydrogen bonds. In the assays performed with the Taq polymerase, the pH dependence (in the range of 6.0-9.0) and the temperature dependence of the arrests were determined. As the pH was lowered, the arrests in the d(TC)27 tract were enhanced, in line with the expected properties of d(TC)i.d(GA)i.d(TC)i triplexes. The arrests in the d(GA)27 tract were enhanced by an increase in the pH. At pH 7.2 the arrests in the d(GA)27 tract persisted up to 80 degrees C, whereas the arrests in the d(TC)27 tract were eliminated at 50 degrees C; these results presumably reflect the relative stabilities of the two triplexes mentioned above at this physiological pH value and could be biologically significant.

Topics: Adenine; Base Sequence; DNA Replication; DNA-Binding Proteins; DNA-Directed DNA Polymerase; DNA, Single-Stranded; Escherichia coli; Guanine; Hydrogen Bonding; Kinetics; Models, Structural; Molecular Sequence Data; Nucleic Acid Conformation; Polydeoxyribonucleotides; Polymerase Chain Reaction; Templates, Genetic; Thermodynamics