Hoogsteen Research Articles

Fluorescence Detection of Single‐Nucleotide Polymorphism with Single‐Strand Triplex‐Forming DNA Probes

Triple-helix-forming oligonucleotides (TFOs) are widespread in the genome and have been found in regulatory regions, especially in promoter zones and recombination hotspots of DNA. To specifically detect these polypurine sequences, we designed and synthesized two dual pyrene-labeled single-strand oligonucleotide probes (TFO-FPs) consisting of recognition, linker, and detection sequences. The hybridization processes of TFO-FPs with target polypurine oligonucleotides involve both Watson-Crick and Hoogsteen base-pairings. Through double sensing of oligonucleotide sequences, single mutations of target oligonucleotides are detected by monitoring changes in pyrene fluorescence. The high specificities of the probes are maintained over a wide temperature range without sacrifice of hybridization kinetics.

The effect of ionization and deprotonation of guanine on the formation of base pairs

The effect of the ionization of the guanine on the formation of base pairs is investigated using the B3LYP method with 6-31++G ∗∗ basis set. The base pairing with neutral DNA base lowers the adiabatic ionization potential of guanine, while the base pairing with protonated DNA base increases the ionization potential of guanine. The proton transfer can occur in the Watson–Crick GC and GA base pairs when an electron is lost. The effect of the deprotonation of the guanine cation on the formation of base pairs is also explored. From an energy point of view, the ionization of guanine cannot result in the Watson–Crick mispair formation, but the deprotonation from the guanine cation is in favor of the combination of the G(−H) and guanine. In the Hoogsteen base pairs, the ionization of guanine slightly affects the interaction mode, but it may result in the dissociation of the Hoogsteen base pairs if the DNA bases pairing with guanine are the protonated A, T and C. The deprotonation from guanine cation slightly affects the base pairing energies of the Hoogsteen base pairs.

The nature of base stacking: a Monte Carlo study

To elucidate the physical origin of the preference of nucleic acid bases for stacking over hydrogen bonding in water, Monte Carlo simulations were performed starting from Watson–Crick structures of the adenine–thymine, adenine–uracil and guanine–cytosine base pairs, as well as from the Hoogsteen adenine–thymine base pair, in clusters comprising 400 and 800 water molecules. The simulations employed a newly implemented Metropolis Monte Carlo algorithm based on the extended cluster approach. All simulations reached stacked structures, confirming that such structures are preferred over the hydrogen-bonded Watson–Crick and Hoogsteen base pairs. The Monte Carlo simulations show the complete transition from hydrogen-bonded base pairs to stacked structures in the Monte Carlo framework. Analysis of the average energies shows that the preference of stacked over hydrogen-bonded structures is due to the increased water–base interaction in these structures. This is corroborated by the increased number of water–base hydrogen bonds in the stacked structures.

4-Aminobenzimidazole–1-Methylthymine: A Model for Investigating Hoogsteen Base-Pairing between Adenine and Thymine

We report the infrared spectrum of the 4-aminobenzimidazole-1-methylthymine (4ABI:1MT) heterodimer, detected by femtosecond multiphoton ionization. Based on calculations of both the harmonic and the anharmonic frequencies, the observed vibrational spectrum is assigned to a structure that mimics the Hoogsteen base pairing of adenine and thymine. A notable observation made in the course of this study is that there is a significant imbalance in the observed strengths of the H-bonds. While the N···H-N bond reveals a large red shift of >700 cm(-1) for the NH stretch frequency, the N-H···O bond is characterized by only a 50 cm(-1) shift. The importance of this observation in the formation of Hoogsteen duplexes by thymine-based oligonucleotides is discussed.

Effects of Protein Binding on DNA G-Quadruplex Structures

The non-canonical G-quadruplex is a unique DNA structure that has received widespread attention because of its potential functions not only in vitro but also in vivo. The G-quadruplex is stabilized by formation of Hoogsteen hydrogen bonds in guanine quartets and involves cation coordination and dehydration. Although a number of proteins have been identified that specifically stabilize and destabilize G-quadruplexes, the effect of the protein binding remains unclear. Here, we review the behaviors of proteins binding to G-quadruplexes. We also discuss analyses of the thermodynamic and binding properties of G-quadruplexes in the complexes formed by histones and thrombins. These studies have helped us to understand the essential features of interaction between G-quadruplexes and proteins

Manipulative Interplay of Two Adozelesin Molecules with d(ATTAAT)2 Achieving Ligand-Stacked Watson–Crick and Hoogsteen Base-Paired Duplex Adducts

Previous structural studies of the cyclopropapyrroloindole (CPI) antitumor antibiotics have shown that these ligands bind covalently edge-on into the minor groove of double-stranded DNA. Reversible covalent modification of the DNA via N3 of adenine occurs in a sequence-specific fashion. Early nuclear magnetic resonance and molecular modeling studies with both mono- and bis-alkylating ligands indicated that the ligands fit tightly within the minor groove, causing little distortion of the helix. In this study, we propose a new binding model for several of the CPI-based analogues, in which the aromatic secondary rings form π-stacked complexes within the minor groove. One of the adducts, formed with adozelesin and the d(ATTAAT)(2) sequence, also demonstrates the ability of these ligands to manipulate the DNA of the binding site, resulting in a Hoogsteen base-paired adduct. Although this type of base pairing has been previously observed with the bisfunctional CPI analogue bizelesin, this is the first time that such an observation has been made with a monoalkylating nondimeric analogue. Together, these results provide a new model for the design of CPI-based antitumor antibiotics, which also has a significant bearing on other structurally related and structurally unrelated minor groove-binding ligands. They indicate the dynamic nature of ligand-DNA interactions, demonstrating both DNA conformational flexibility and the ability of two DNA-bound ligands to interact to form stable covalent modified complexes.

DFT study of DNA sequence dependence at the level of dinucleoside monophosphates

In this work we search for new energy minima of desoxydinucleoside monophosphate complexes with Na-ions (dDMPs) as the minimal fragments of DNA single strand via Density Functional Theory (DFT) computations and summarize our previous findings in this field. The analysis is illustrated by graphs of distribution of sugar-phosphate and glycosyl torsions as well as of sugar puckering for energy minima of various base sequences to elucidate the contributions of intra-strand interactions to DNA structure and polymorphism. Our recent computations revealed that the characteristic features of BI, BII, and AI families of DNA duplex conformations are initially predisposed in the dDMPs and namely in their local energy minima. New calculations and the analysis of our previous results enable us to verify and to draw conclusions about important role of sugar-phosphate backbone in the formation and sequence dependence of DNA duplexes. The optimized conformations of dDMPs with purine–purine and purine–pyrimidine sequences are characterized by substantial superposition of base rings whereas those with pyrimidine–pyrimidine and pyrimidine–purine sequences have small if any superposition. This result reveals itself as a universal rule for energy minima corresponding to all four families of right-handed duplexes with Watson–Crick base pairs (BI, BII, AI and AII). The character of base–base overlap is independent of the degree of variation of each of the backbone torsions, which may vary substantially or only slightly with the nucleoside sequence within a family. Computations on dDMP structures corresponding to duplexes with Hoogsteen pairs, started from the torsion angle values of the duplex, located characteristic energy minima for dApdT and dTpdA representative of this conformation. We observe that the rule of sequence dependence of base superposition does not apply to these systems. Biological consequences of existence of the obtained minima and the implications of the revealed regularities are discussed.

8-Oxoguanosine Switches Modulate the Activity of Alkylated siRNAs by Controlling Steric Effects in the Major versus Minor Grooves

Small interfering double-stranded RNAs have been synthesized bearing one or more base modifications at nucleotide positions 4, 11, and/or 16 in the guide strand. The chemically modified base is an N(2)-alkyl-8-oxo-7,8-dihydroguanine (alkyl = propyl, benzyl) that can alternatively pair in a Watson-Crick sense opposite cytosine (C) or as a Hoogsteen pair opposite adenine (A). Cellular delivery with C opposite led to effective targeting of A-containing but not C-containing mRNA sequences in a dual luciferase assay with RNA interference levels that were generally as good as or better than unmodified sequences. The higher activity is ascribed to an inhibitory effect of the alkyl group projecting into the minor groove of double-stranded RNA preventing off-target binding to proteins such as PKR (RNA-activated protein kinase).

Designed Nucleotide Binding Motifs

Gaps in the central strand of oligonucleotide triplexes bind nucleoside phosphates tightly. Watson-Crick and Hoogsteen base pairing as design principle yield motifs with high affinity for nucleoside phosphates with A or G as nucleobase, including ATP. The second messenger 3',5'-cAMP is bound with nanomolar affinity. A designed DNA motif accommodates seven nucleotides at a time. The design was implemented for both DNA and RNA.

Localization and anharmonicity of the vibrational modes for GC Watson–Crick and Hoogsteen base pairs

We present an ab initio study of the vibrational properties of cytosine and guanine in the Watson-Crick and Hoogsteen base pair configurations. The results are obtained by using two different implementations of the DFT method. We assign the vibrational frequencies to cytosine or to guanine using the vibrational density of states. Next, we investigate the importance of anharmonic corrections for the vibrational modes. In particular, the unusual anharmonic effect of the H(+) vibration in the case of the Hoogsteen base pair configuration is discussed.

Transient Hoogsteen base pairs in canonical duplex DNA

Sequence-directed variations in the canonical DNA double helix structure that retain Watson-Crick base-pairing play important roles in DNA recognition, topology, and nucleosome positioning. By using nuclear magnetic resonance relaxation dispersion spectroscopy in concert with steered molecular dynamics simulations, we have observed transient sequence-specific excursions away from Watson-Crick base-pairing at CA and TA steps inside canonical duplex DNA towards low-populated and short-lived A•T and G•C Hoogsteen base-pairs. The observation of Hoogsteen base-pairs in DNA duplexes specifically bound to transcription factors and in damaged DNA sites implies that the DNA double helix intrinsically codes for excited state Hoogsteen base-pairs as a means of expanding its structural complexity beyond that which can be achieved based on Watson-Crick base-pairing. The methods presented here provide a new route for characterizing transient low-populated nucleic acid structures, which we predict will be abundant in the genome and constitute a second transient layer of the genetic code.

Molecular Recognition of the Antiretroviral Drug Abacavir: Towards the Development of a Novel Carbazole-Based Fluorosensor

Due to their optical and electro-conductive attributes, carbazole derivatives are interesting materials for a large range of biosensor applications. In this study, we present the synthesis routes and fluorescence evaluation of newly designed carbazole fluorosensors that, by modification with uracil, have a special affinity for antiretroviral drugs via either Watson–Crick or Hoogsteen base pairing. To an N-octylcarbazole-uracil compound, four different groups were attached, namely thiophene, furane, ethylenedioxythiophene, and another uracil; yielding four different derivatives. Photophysical properties of these newly obtained derivatives are described, as are their interactions with the reverse transcriptase inhibitors such as abacavir, zidovudine, lamivudine and didanosine. The influence of each analyte on biosensor fluorescence was assessed on the basis of the Stern–Volmer equation and represented by Stern–Volmer constants. Consequently we have demonstrated that these structures based on carbazole, with a uracil group, may be successfully incorporated into alternative carbazole derivatives to form biosensors for the molecular recognition of antiretroviral drugs.

Thermodynamic stability of Hoogsteen and Watson–Crick base pairs in the presence of histone H3-mimicking peptide

We found that Hoogsteen base pairs were stabilized by molecular crowding and a histone H3-mimicking peptide, which was not observed for Watson-Crick base pairs. Our findings demonstrate that the type of DNA base pair is critical for the interaction between DNA and histones.

DNA breathes Hoogsteen

A recent claim is discussed that Watson-Crick pairs in the naked duplex DNA spontaneously flip into Hoogsteen pairs under ordinary conditions. The claim is considered within the historical retrospective and is put into the broader context of DNA biophysics.

Structural Basis for Proficient Incorporation of dTTP Opposite O6-Methylguanine by Human DNA Polymerase ι

O(6)-methylguanine (O(6)-methylG) is highly mutagenic and is commonly found in DNA exposed to methylating agents, even physiological ones (e.g. S-adenosylmethionine). The efficiency of a truncated, catalytic DNA polymerase ι core enzyme was determined for nucleoside triphosphate incorporation opposite O(6)-methylG, using steady-state kinetic analyses. The results presented here corroborate previous work from this laboratory using full-length pol ι, which showed that dTTP incorporation occurs with high efficiency opposite O(6)-methylG. Misincorporation of dTTP opposite O(6)-methylG occurred with ∼6-fold higher efficiency than incorporation of dCTP. Crystal structures of the truncated form of pol ι with O(6)-methylG as the template base and incoming dCTP or dTTP were solved and showed that O(6)-methylG is rotated into the syn conformation in the pol ι active site and that dTTP misincorporation by pol ι is the result of Hoogsteen base pairing with the adduct. Both dCTP and dTTP base paired with the Hoogsteen edge of O(6)-methylG. A single, short hydrogen bond formed between the N3 atom of dTTP and the N7 atom of O(6)-methylG. Protonation of the N3 atom of dCTP and bifurcation of the N3 hydrogen between the N7 and O(6) atoms of O(6)-methylG allow base pairing of the lesion with dCTP. We conclude that differences in the Hoogsteen hydrogen bonding between nucleotides is the main factor in the preferential selectivity of dTTP opposite O(6)-methylG by human pol ι, in contrast to the mispairing modes observed previously for O(6)-methylG in the structures of the model DNA polymerases Sulfolobus solfataricus Dpo4 and Bacillus stearothermophilus DNA polymerase I.

An indole-linked C8-deoxyguanosine nucleoside acts as a fluorescent reporter of Watson–Crick versus Hoogsteen base pairing

Pyrrole- and indole-linked C(8)-deoxyguanosine nucleosides act as fluorescent reporters of H-bonding specificity. Their fluorescence is quenched upon Watson-Crick H-bonding to dC, while Hoogsteen H-bonding to G enhances emission intensity. The indole-linked probe is ∼ 10-fold brighter and shows promise as a fluorescent reporter of Hoogsteen base pairing.

Using triplex-forming oligonucleotide probes for the reagentless, electrochemical detection of double-stranded DNA.

We report a reagentless, electrochemical sensor for the detection of double-stranded DNA targets that employs triplex-forming oligonucleotides (TFOs) as its recognition element. These sensors are based on redox-tagged TFO probes strongly chemisorbed onto an interrogating gold electrode. Upon the addition of the relevant double-stranded DNA target, the probe forms a rigid triplex structure via reverse Hoogsteen base pairing in the major groove. The formation of the triplex impedes contact between the probe's redox moiety and the interrogating electrode, thus signaling the presence of the target. We first demonstrated the proof of principle of this approach by using a well-characterized 22-base polypurine TFO sequence that readily detects a synthetic, double-stranded DNA target. We then confirmed the generalizability of our platform with a second probe, a 19-base polypyrimidine TFO sequence that targets a polypurine tract (PPT) sequence conserved in all HIV-1 strains. Both sensors rapidly and specifically detect their double-stranded DNA targets at concentrations as low as ~10 nM and are selective enough to be employed directly in complex sample matrices such as blood serum. Moreover, to demonstrate real-world applicability of this new sensor platform, we have successfully detected unpurified, double-stranded PCR amplicons containing the relevant conserved HIV-1 sequence.

Effect of Watson−Crick and Hoogsteen Base Pairing on the Conformational Stability of C8-Phenoxyl-2′-deoxyguanosine Adducts

Bulky DNA addition products (adducts) formed through attack at the C8 site of guanine can adopt the syn orientation about the glycosidic bond due to changes in conformational stability or hydrogen-bonding preferences directly arising from the bulky group. Indeed, the bulky substituent may improve the stability of (non-native) Hoogsteen pairs. Therefore, such adducts often result in mutations upon DNA replication. This work examines the hydrogen-bonded pairs between the Watson-Crick and Hoogsteen faces of the ortho or para C8-phenoxyl-2'-deoxyguanosine adduct and each natural (undamaged) nucleobase with the goal to clarify the conformational preference of this type of damage, as well as provide insight into the likelihood of subsequent mutation events. B3LYP/6-311+G(2df,p)//B3LYP/6-31G(d) hydrogen-bond strengths were determined using both nucleobase and nucleoside models for adduct pairs, as well as the corresponding complexes involving natural 2'-deoxyguanosine. In addition to the magnitude of the binding strengths, the R(C1'···C1') distances and ∠(N9C1'C1') angles, as well as the degree of propeller-twist and buckle distortions, were carefully compared to the values observed in natural DNA strands. Due to structural changes in the adduct monomer upon inclusion of the sugar moiety, the monomer deformation energy significantly affects the relative hydrogen-bond strengths calculated with the nucleobase and nucleoside models. Therefore, we recommend the use of at least a nucleoside model to accurately evaluate hydrogen-bond strengths of base pairs involving flexible, bulky nucleobase adducts. Our results also emphasize the importance of considering both the magnitude of the hydrogen-bond strength and the structure of the base pair when predicting the preferential binding patterns of nucleobases. Using our best models, we conclude that the Watson-Crick face of the ortho phenoxyl adduct forms significantly more stable complexes than the Hoogsteen face, which implies that the anti orientation of the damaged base will be favored by hydrogen bonding in DNA helices. Additionally, regardless of the hydrogen-bonding face involved, cytosine forms the most stable base pair with the ortho adduct, which implies that misincorporation due to this type of damage is unlikely. Similarly, cytosine is the preferred binding partner for the Watson-Crick face of the para adduct. However, Hoogsteen interactions with the para adduct are stronger than those with natural 2'-deoxyguanosine or the ortho adduct, and this form of damage binds with nearly equal stability to both cytosine and guanine in the Hoogsteen orientation. Therefore, the para adduct may adopt multiple orientations in DNA helices and potentially cause mutations by forming pairs with different natural bases. Models of oligonucleotide duplexes must be used in future work to further evaluate other factors (stacking, major groove contacts) that may influence the conformation and binding preference of these adducts in DNA helices.

Direct Voltammetric Analysis of DNA Modified with Enzymatically Incorporated 7-Deazapurines

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.

Triplexes with 8‐Aza‐2′‐Deoxyisoguanosine Replacing Protonated dC: Probing Third Strand Stability with a Fluorescent Nucleobase Targeting Duplex DNA

The fluorescent 8-aza-2'-deoxyisoguanosine (4) as well as the parent 2'-deoxyisoguanosine (1) were used as protonated dCH(+) surrogates in the third strand of oligonucleotide triplexes. Stable triplexes were formed by Hoogsteen base pairing. In contrast to dC, triplexes containing nucleoside 1 or 4 in place of dCH(+) are already formed under neutral conditions or even at alkaline pH values. Triplex melting can be monitored separately from duplex dissociation in cases in which the third strand contains the fluorescent nucleoside 4. Third-strand binding of oligonucleotides with 4, opposite to dG, was selective as demonstrated by hybridisation experiments studying mismatch discrimination. Third-strand binding is more efficient when the stability of the DNA duplex is reduced by mismatches, giving third-strand binding more flexibility.