Structural basis of DNA folding and recognition in an AMP-DNA aptamer complex: distinct architectures but common recognition motifs for DNA and RNA aptamers complexed to AMP

Background: Structural studies by nuclear magnetic resonance (NMR) of RNA and DNA aptamer complexes identified through in vitro selection and amplification have provided a wealth of information on RNA and DNA tertiary structure and molecular recognition in solution. The RNA and DNA aptamers that target ATP (and AMP)' with micromolar affinity exhibit distinct binding site sequences and secondary structures. We report below on the tertiary structure of the AMP-DNA aptamer complex in solution and compare it with the previously reported tertiary structure of the AMP-RNA aptamer complex in solution.Results: The solution structure of the AMP-DNA aptamer complex shows, surprisingly, that two AMP molecules are intercalated at adjacent sites within a rectangular widened minor groove. Complex formation involves adaptive binding where the asymmetric internal bubble of the free DNA aptamer zippers up through formation of a continuous six-base mismatch segment which includes a pair of adjacent three-base platforms. The AMP molecules pair through their Watson-Crick edges with the minor groove edges of guanine residues. These recognition G·A mismatches are flanked by sheared G·A and reversed Hoogsteen G·G mismatch pairs.Conclusions: The AMP-DNA aptamer and AMP-RNA aptamer complexes have distinct tertiary structures and binding stoichiometries. Nevertheless, both complexes have similar structural features and recognition alignments in their binding pockets. Specifically, AMP targets both DNA and RNA aptamers by intercalating between purine bases and through identical G·A mismatch formation. The recognition G·A mismatch stacks with a reversed Hoogsteen G·G mismatch in one direction and with an adenine base in the other direction in both complexes. It is striking that DNA and RNA aptamers selected independently from libraries of 10¹⁴ molecules in each case utilize identical mismatch alignments for molecular recognition with micromolar affinity within binding-site pockets containing common structural elements.     

Long-range oxidation of guanine by Ru(III) in duplex DNA

Background: Theoretical and experimental studies have demonstrated that 5′-GG-3′ sequences in DNA are ‘hot spots’ for oxidative damage, but few studies have definitively addressed whether oxidative damage to DNA may arise from a distance via long-range charge migration. Towards this end, we have prepared tethered ruthenium (Ru)-oligonucleotide duplexes and used a flash—quench strategy to demonstrate long-range charge transport through the DNA double helix.Results: DNA assemblies containing a tethered Ru(II) intercalator have been synthesized. Ru(III), generated in situ in the presence of externally bound electron-transfer quenchers, promotes base damage selectively at the 5′-G of a 5′-GG-3′ doublet located ∼ 37 Å from the binding site of the oxidant. In the absence of a guanine doublet, oxidative damage occurs equally at all guanine bases in the strand. Oxidative damage is also observed at long range for guanine in a G·A mismatch but not in a G·T mismatch.Conclusions: The present study expands the scope of long-range electron-transfer chemistry in terms of experiments, applications, and possible reactions within the cell. Here we demonstrate oxidative damage to DNA occurring with a high quantum yield over a distance of ∼37 Å using a ground-state oxidant. These results point to the equilibration of the radical across the DNA duplex to the sites of lowest energy. In addition, this charge migration is sensitive to the intervening π-stack formed by DNA base pairs and hence may be useful for the detection of mismatches.     

Encapsulating streptomycin within a small 40-mer RNA

We describe a 2.9 A X-ray structure of a complex between the aminocyclitol antibiotic streptomycin and an in vitro selected RNA aptamer, solved using the anomalous diffraction properties of Ba cations. The RNA aptamer, which contains two asymmetric internal loops, adopts a distinct cation-stabilized fold involving a series of S-shaped backbone turns anchored by canonical and noncanonical pairs and triples. The streptomycin streptose ring is encapsulated by stacked arrays of bases from both loops at the elbow of the L-shaped RNA architecture. Specificity is defined by direct hydrogen bonds between all streptose functional groups and base edges that line the inner walls of the cylindrical binding pocket. By contrast, the majority of intermolecular interactions involve contacts to backbone phosphates in the published structure of streptomycin bound to the 16S rRNA.     

Structural basis for discriminative regulation of gene expression by adenine- and guanine-sensing mRNAs

Metabolite-sensing mRNAs, or "riboswitches," specifically interact with small ligands and direct expression of the genes involved in their metabolism. Riboswitches contain sensing "aptamer" modules, capable of ligand-induced structural changes, and downstream regions, harboring expression-controlling elements. We report the crystal structures of the add A-riboswitch and xpt G-riboswitch aptamer modules that distinguish between bound adenine and guanine with exquisite specificity and modulate expression of two different sets of genes. The riboswitches form tuning fork-like architectures, in which the prongs are held in parallel through hairpin loop interactions, and the internal bubble zippers up to form the purine binding pocket. The bound purines are held by hydrogen bonding interactions involving conserved nucleotides along their entire periphery. Recognition specificity is associated with Watson-Crick pairing of the encapsulated adenine and guanine ligands with uridine and cytosine, respectively.     

Bulged Guanine is Uniquely Sensitive to Damage Caused by Visible‐Light Irradiation of Ethidium Bound to DNA: A Possible Role in Mutagenesis

The interaction of ethidium bromide (=3,8‐diamino‐5‐ethyl‐6‐phenylphenanthridinium bromide; EB) with a series of duplex DNA oligomers having single‐base bulges and single‐base mis‐pairs was investigated (Fig. 1). Physical and spectroscopic analysis reveals no definitive evidence for selective binding of EB at the bulge or mis‐pair. However, irradiation of the bound EB with VIS light leads to lesions in the DNA selectively in the sequence having a bulged guanine. This reaction is attributed to the formation of an exciplex between the lowest excited singlet state of the EB and the bulged guanine. The exciplex is trapped by H₂O, which initiates a sequence of reactions that lead to piperidine‐requiring strand cleavage at this site. Significantly, the damaged bulged guanine is not recognized by E. coli formamidopyrimidine glycosylase (Fpg), which is part of a base‐excision repair system for oxidative damage to DNA. Thus, DNA containing a bulged guanine and having a bound intercalator may be irreparably damaged by exposure to VIS light, even though normal duplex DNA is relatively inert under these conditions.     

Oxidation of guanines in the iron-responsive element RNA: similar structures from chemical modification and recent NMR studies

Background: The translation or stability of the mRNAs from ferritin, m-aconitase, erythroid aminoevulinate synthase and the transferrin receptor is controlled by the binding of two iron regulatory proteins to a family of hairpin-forming RNA sequences called iron-responsive elements (IREs). The determination of higher-solution nuclear magnetic resonance (NMR) structures of IRE variants suggests an unusual hexaloop structure, leading to an intra-loop G-C base pair and a highly exposed loop guanine, and a special internal loop/bulge in the ferritin IRE involving a shift in base pairing not predicted with standard algorithms.Results: Cleavage of synthetic 55- and 30-mer RNA oligonucleotides corresponding to the ferritin IRE with complexes based on oxoruthenium(IV) shows enhanced reactivity at a hexaloop guanine and at a guanine adjacent to the internal loop/bulge with strong protection at a guanine in the internal loop/bulge. These results are consistent with the recent NMR structures. The synthetic 55-mer RNA binds the iron-regulatory protein from rabbit reticulocyte lysates. The DNA analogs of the 55- and 30-mers do not show the same reactivity pattern.Conclusions: The chemical reactivity of the guanines in the ferritin IRE towards oxoruthenium(IV) supports the published NMR structures and the known oxidation chemistry of the metal complexes, The results constitute progress towards developing stand-alone chemical nucleases that reveal significant structural properties and provide results that can ultimately be used to constrain molecular modeling.     

Four-stranded DNA structures can be stabilized by two different types of minor groove G:C:G:C tetrads

Four-stranded nucleic acid structures are central to many processes in biology and in supramolecular chemistry. It has been shown recently that four-stranded DNA structures are not only limited to the classical guanine quadruplex but also can be formed by tetrads resulting from the association of Watson-Crick base pairs. Such an association may occur through the minor or the major groove side of the base pairs. Structures stabilized by minor groove tetrads present distinctive features, clearly different from the canonical guanine quadruplex, making these quadruplexes a unique structural motif. Within our efforts to study the sequence requirements for the formation of this unusual DNA motif, we have determined the solution structure of the cyclic oligonucleotide dpCCGTCCGT by two-dimensional NMR spectroscopy and restrained molecular dynamics. This molecule self-associates, forming a symmetric dimer stabilized by two G:C:G:C tetrads with intermolecular G-C base pairs. Interestingly, although the overall three-dimensional structure is similar to that found in other cyclic and linear oligonucleotides of related sequences, the tetrads that stabilize the structure of dpCCGTCCGT are different to other minor groove G:C:G:C tetrads found earlier. Whereas in previous cases the G-C base pairs aligned directly, in this new tetrad the relative position of the two base pairs is slipped along the axis defined by the base pairs. This is the first time that a quadruplex structure entirely stabilized by slipped minor groove G:C:G:C tetrads is observed in solution or in the solid state. However, an analogous arrangement of G-C base pairs occurs between the terminal residues of contiguous duplexes in some DNA crystals. This structural polymorphism between minor groove GC tetrads may be important in stabilization of higher order DNA structures.     

Selectivity of quadruplex DNA stationary phases toward amino acids in homodipeptides and alanyl dipeptides

Series of dipeptides, including homodipeptides and alanyl dipeptides, were separated using quadruplex (G-quartet) DNA stationary phases in open-tubular capillary electrochromatography (OTCEC). The stationary phases were constructed by covalently attaching the DNA oligonucleotides to the inner capillary surface. Three different G-quartet forming oligonucleotides were investigated: the two-plane G-quartet forming thrombin-binding aptamer, the four-plane analogue of the thrombin-binding aptamer, and a two-plane oligonucleotide identical to the thrombin-binding aptamer except for the replacement of the guanine by thymine in the central bridging loop of the G-quartet structure. Results were compared with results obtained using capillary electrophoresis on a bare capillary and OTCEC using an oligonucleotide with the same base composition as the thrombin-binding aptamer but in a different sequence that does not allow G-quartet formation as the stationary phase.     

RNA architecture dictates the conformations of a bound peptide.

BACKGROUND: The biological function of several viral and bacteriophage proteins, and their arginine-rich subdomains, involves RNA-mediated interactions. It has been shown recently that bound peptides adopt either beta-hairpin or alpha-helical conformations in viral and phage peptide-RNA complexes. We have compared the structures of the arginine-rich peptide domain of HIV-1 Rev bound to two RNA aptamers to determine whether RNA architecture can dictate the conformations of a bound peptide. RESULTS: The core-binding segment of the HIV-1 Rev peptide class II RNA aptamer complex spans the two-base bulge and hairpin loop of the bound RNA and the carboxy-terminal segment of the bound peptide. The bound peptide is anchored in place by backbone and sidechain intermolecular hydrogen bonding and van der Waals stacking interactions. One of the bulge bases participates in U*(A*U) base triple formation, whereas the other is looped out and flaps over the bound peptide in the complex. The seven-residue hairpin loop is closed by a sheared G*A mismatch pair with several pyrimidines looped out of the hairpin fold. CONCLUSIONS: Our structural studies establish that RNA architecture dictates whether the same HIV-1 Rev peptide folds into an extended or alpha-helical conformation on complex formation. Arginine-rich peptides can therefore adapt distinct secondary folds to complement the tertiary folds of their RNA targets. This contrasts with protein-RNA complexes in which elements of RNA secondary structure adapt to fit within the tertiary folds of their protein targets.     

Silver Ions in Non‐canonical DNA Base Pairs: Metal‐Mediated Mismatch Stabilization of 2′‐Deoxyadenosine and 7‐Deazapurine Derivatives with 2′‐Deoxycytidine and 2′‐Deoxyguanosine

Novel silver‐mediated dA?dC, dA*?dC, and dA*?dG base pairs were formed in a natural DNA double helix environment (dA* denotes 7‐deaza‐dA, 7‐deaza‐7‐iodo‐dA, and 7‐cyclopropyl‐7‐deaza‐dA). 7‐Deazapurine nucleosides enforce silver ion binding and direct metal‐mediated base pair formation to their Watson–Crick face. New phosphoramidites were prepared from 7‐deaza‐dA, 7‐deaza‐7‐iodo‐dA, and 7‐cyclopropyl‐7‐deaza‐dA, which contain labile isobutyryl protecting groups. Solid‐phase synthesis furnished oligonucleotides that contain mismatches in near central positions. Increased thermal stabilities (higher T_m values) were observed for oligonucleotide duplexes with non‐canonical dA*?dC and dA?dC pairs in the presence of silver ions. The stability of the silver‐mediated base pairs was pH dependent. Silver ion binding was not observed for the dA?dG mismatch but took place when mismatches were formed between 7‐deazaadenine and guanine. The specific binding of silver ions was confirmed by stoichiometric UV titration experiments, which proved that one silver ion is captured by one mismatch. The stability increase of canonical DNA mismatches might have an impact on cellular DNA repair.     

Triad base pairs containing fluorene unit for quencher-free SNP typing

We designed an A-selective fluorescent DNA probe, U^FL, which bears a 2-ethynylfluorene moiety covalently attached to the base dU, and incorporated it into a central position in the hairpin loop. We describe photophysical studies of triad base pairs containing an U^FL DNA probe and their application to single-nucleotide polymorphism (SNP) typing using novel quencher-free molecular beacons (MBs). The drastic changes in the fluorescence properties that arise upon changing the nature of the complementary base and FBs suggest that these triad base pairs are the key elements for quencher-free SNP typing.     

On the pairing rules for recognition in the minor groove of DNA by pyrrole-imidazole polyamides

Background: Cell-permeable small molecules that target predetermined DNA sequences with high affinity and specificity have the potential to control gene expression. A binary code has been developed to correlate DNA sequence with side-by-side pairings between N-methylpyrrole (Py) and N-methylimidazole (lm) carboxamides in the DNA minor groove. We set out to determine the relative energetics of pairings of Im/Py, Py/Im, Im/Im, and Py/Py for targeting G·C and A·T base pairs. A key specificity issue, which has not been previously addressed, is whether an Im/Im pair is energetically equivalent to an Im/Py pair for targeting G·C base pairs.Results: Equilibrium association constants were determined at two five-base-pair sites for a series of four six-ring hairpin polyamides, in order to test the relative energetics of the four aromatic amino-acid pairings opposite G·C and A·T base pairs in the central position. We observed that a G·C base pair was effectively targeted with Im/Py but not Py/Im, Py/Py, or Im/Im. The A·T base pair was effectively targeted with Py/Py but not Im/Py, Py/Im, or Im/Im.Conclusions: An Im/Im pairing is energetically disfavored for the recognition of both A·T and G·C. This specificity will create important limitations on undesirable slipped motifs that are available for unlinked dimers in the minor groove. Baseline energetic parameters will thus be created which, using the predictability of the current pairing rules for specific molecular recognition of double-helical DNA, will guide further second-generation polyamide design for DNA recognition.     

Influence of intervening mismatches on long-range guanine oxidation in DNA duplexes

A systematic investigation of the efficiency of oxidative damage at guanine residues through long-range charge transport was carried out as a function of intervening base mismatches. A series of DNA oligonucleotides were synthesized that incorporate a ruthenium intercalator linked covalently to the 5' terminus of one strand and containing two 5'-GG-3' sites in the complementary strand. Single base mismatches were introduced between the two guanine doublet steps, and the efficiency of transport through the mismatches was determined through measurements of the ratio of oxidative damage at the guanine doublets distal versus proximal to the intercalated ruthenium oxidant. Differing relative extents of guanine oxidation were observed for the different mismatches. The damage ratio of oxidation at the distal versus proximal site for the duplexes containing different mismatches varies in the order GC approximately GG approximately GT approximately GA > AA > CC approximately TT approximately CA approximately CT. For all assemblies, damage found with the Delta-Ru diastereomer was found to be greater than with the Lambda-diastereomer. The extent of distal/proximal guanine oxidation in different mismatch-containing duplexes was compared with the helical stability of the duplexes, electrochemical data for intercalator reduction on different mismatch-containing DNA films, and base-pair lifetimes for oligomers containing the different mismatches derived from 1H NMR measurements of the imino proton exchange rates. While a clear correlation is evident both with helix stability and electrochemical data monitoring reduction of an intercalator through DNA films, damage ratios correlate most closely with base-pair lifetimes. Competitive hole trapping at the mismatch site does not appear to be a key factor governing the efficiency of transport through the mismatch. These results underscore the importance of base dynamics in modulating long-range charge transport through the DNA base-pair stack.     

Rapid radical formation by DNA charge transport through sequences lacking intervening guanines

Using the flash-quench technique to probe DNA charge transport in assemblies containing a tethered ruthenium intercalator, the kinetics and yield of methylindole radical formation as a function of DNA sequence were studied by laser spectroscopy and biochemical methods. In these assemblies, the methylindole moiety serves as an artificial base of low oxidation potential. Hole injection and subsequent formation of the methylindole radical cation were observed at a distance of over 30 A at rates >/=107 s-1 in assemblies containing no guanine bases intervening the ruthenium intercalator and GMG oxidation site. Radical yield was, however, strikingly sensitive to an intervening base mismatch; no significant methylindole radical formation was evident with an intervening AA mismatch. Also critical is the sequence at the injection site; this sequence determines initial hole localization and hence the probability of hole propagation. With guanine rather than inosine near the site of hole injection, decreased yields of radicals and long-range oxidative damage are observed. The presence of the low-energy guanine site in this case serves to localize the hole and therefore diminish charge transport through the base pair stack.     

A Three-dimensional Model of the Human Immunodeficiency Virus Type 1 Integration Complex

Summary While the general features of HIV-1 integrase function are understood, there is still uncertainty about the composition of the integration complex and how integrase interacts with viral and host DNA. We propose an improved model of the integration complex based on current experimental evidence including a comparison with the homologous Tn5 transposase containing bound DNA and an analysis of DNA binding sites using Goodford’s GRID. Our model comprises a pair of integrase dimers, two strands of DNA to represent the viral DNA ends and a strand of bent DNA representing the host chromosome. In our model, the terminal four base pairs of each of the viral DNA strands interact with the integrase dimer providing the active site, while bases one turn away interact with a flexible loop (residues 186–194) on the second integrase dimer. We propose that residues E152, Q148 and K156 are involved in the specific recognition of the conserved CA dinucleotide and that the active site mobile loop (residues 140–149) stabilises the integration complex by acting as a barrier to separate the two viral DNA ends. In addition, the residues responsible for DNA binding in our model show a high level of amino acid conservation.     

5-Aza-7-deazaguanine–Isoguanine and Guanine–Isoguanine Base Pairs in Watson–Crick DNA: The Impact of Purine Tracts,Clickable Dendritic Side Chains,and Pyrene Adducts

The Watson–Crick coding system depends on the molecular recognition of complementary purine and pyrimidine bases. Now, the construction of hybrid DNAs with Watson–Crick and purine–purine base pairs decorated with dendritic side chains was performed. Oligonucleotides with single and multiple incorporations of 5-aza-7-deaza-2′-deoxyguanosine, its tripropargylamine derivative, and 2′-deoxyisoguanosine were synthesized. Duplex stability decreased if single modified purine–purine base pairs were inserted, but increased if pyrene residues were introduced by click chemistry. A growing number of consecutive 5-aza-7-deazaguanine–isoguanine base pairs led to strong stepwise duplex stabilization, a phenomenon not observed for the guanine–isoguanine base pair. Spacious residues are well accommodated in the large groove of purine–purine DNA tracts. Changes to the global helical structure monitored by circular dichroism spectroscopy show the impact of functionalization to the global double-helix structure. This study explores new areas of molecular recognition realized by purine base pairs that are complementary in hydrogen bonding, but not in size, relative to canonical pairs.     

Bi-stranded, multisite replication of a base pair between difluorotoluene and adenine: confirmation by ‘inverse’ sequencing

Background: The nonpolar nucleoside of difluorotoluene (F) was previously found to behave similarly to thymidine in single-site deoxynucleoside triphosphate (dNTP) insertion experiments with the Klenow fragment (KF) of DNA polymerase I. Further study was needed, first to see whether F-A base pairs could be replicated in more than one sequence context; second to investigate whether specific base pair replication occurs in the presence of four dNTPs; and third to confirm the presence of F in a replicated DNA strand.Results: A primer bound to a template strand containing eight F residues was extended by KF using the four natural dNTPs at 20 μM. Similarly, the complement (containing eight adenines) was extended using dATP, dGTP, dCTP and dFTP. Comparison of the new strands to authentic strands using standard and ‘inverse’ chemical sequencing showed identical composition within ± 5%.Conclusions: The results confirm that F in a template strand encodes the insertion of dATP and that adenine in a template encodes the insertion of dFTP with good specificity in at least six different nearest neighbor contexts. The results confirm that analog F behaves similarly to thymidine despite its poor hydrogen-bonding ability.     

Visualisation of inter loop tertiary base pairing and stacking interactions in an unusual slipped loop DNA structure

The conformation of an unusual slipped loop DNA structure exhibited by the sequence d(GAATTCCCGAATTC)₂ is determined using a combination of geometrical and molecular mechanics methods. This sequence is known to form a B-DNA-like duplex with the central non-complementary cytosines extruded into single stranded loop regions. The unusual feature is that the interior guanine does not pair with the cytosine across, instead, it pairs with the cytosine upstream by skipping two cytosines, leading to a slipped loop DNA structure with the loops staggered by two base pairs. The two loops, despite being very small, can fold across minor or major groove symmetrically or asymmetrically disposed, with one of the loop bases partially blocking the major or minor groove. Most interestingly, for certain conformations, the loop bases approach one another at close proximity so as to engage even in base pairing as well as base stacking interactions across the major groove. While such pairing and stacking are common in the tertiary folds of RNA, this is the first time that such an interaction is visualized in a DNA. This observation demonstrates that a W-C pair can readily be accomplished in a typical slipped loop structure postulated for DNA. Such tertiary loop interaction may prevent access to regulatory proteins across the major groove of the duplex DNA, thus providing a structure-function relation for the occurrence of slipped loop structure in DNA. Contribution no. 839 from this department     

Watson–Crick versus imidazopyridopyrimidine base pairs: theoretical study on differences in stability and hydrogen bonding strength

Matsuda and coworkers demonstrated that imidazopyridopyrimidine nucelobases (N ^N , O ^O , N ^O , tO ^O , and O ^N ) can mimic Watson–Crick nucleobase in forming H-bonds in DNA double helix. In the present study, we address the question about the strengths of the H-bonds in imidazopyridopyrimidine base pairs compared to those in Watson–Crick ones by focusing particularly on the nature of these interactions. Optimized structures of imidazopyridopyrimidine, imidazopyridopyrimidine–Watson–Crick, and Watson–Crick base pairs are obtained at the DFTB3LYP/6-311++G (d,p). The nature and strength of the intramolecular H-bonds in these base pairs have been investigated based on natural bond orbital (NBO method) to consider the effect of charge transfer, “atoms-in-molecules” (AIM) topological parameters, and decomposition of the interaction energies using the energy decomposition analysis (EDA). These investigations imply that N ^N –O ^O and N ^O –O ^N can form base pairs with four H-bonds (most stable than those of Watson–Crick base pairs) when they incorporated into DNA double helix. Furthermore, it can be deduced that O ^N and N ^N nucleobases form energetically more favorable pairs with adenine and guanine than the normal Watson–Crick counter parts. These results can be helpful for the stabilization and regulation of a variety of new base-pairing motif of DNA structures.     

The role of terminal tyrosine residues in the formation of tripeptide nanotubes: a crystallographic insight

Terminally protected acyclic tripeptides containing tyrosine residues at both termini self-assemble into nanotubes in crystals through various non-covalent interactions including intermolecular hydrogen bonds. The nanotube has an average internal diameter of 5 Å (0.5 nm) and the tubular ensemble is developed through the hydrogen-bonded phenolic-OH side chains of tyrosine (Tyr) residues [Org. Lett.2004, 6, 4463]. We have synthesized and studied several tripeptides 3-6 to probe the role of tyrosine residues in nanotube structure formation. These peptides either have only one Tyr residue at N- or C-termini or they have one or two terminally located phenylalanine (Phe) residues. These tripeptides failed to form any kind of nanotubular structure in the solid state. Single crystal X-ray diffraction studies of these peptides 3-6 clearly demonstrate that substitution of any one of the terminal Tyr residues in the Boc-Tyr-X-Tyr-OMe (X=Val or Ile) sequence disrupts the formation of the nanotubular structure indicating that the presence of two terminally located Tyr residues is vital for nanotube formation.