Isostable DNA

The high fidelity detection of multiple DNA sequences in multiplex assays calls for duplexes whose stability is independent of sequence (isostable DNA), forming under universally stringent conditions. Nature did not evolve DNA to form isostable duplexes. Here we report how probe strands can be modified so that an all-A/T target strand is bound with the same or slightly higher affinity than the corresponding all-G/C strand with the same sequence of purines and pyrimidines. We refer to these probes that feature covalently attached ligands as "decorated nucleic acids". Caps, intercalators, and locks were used to stabilize A/T duplexes, and N4-ethylcytosine residues were employed to tune down the stability of G/C duplexes without significantly affecting base pairing selectivity. Near-isostability was demonstrated in solution and on microarrays of high and low density. Further, it is shown that hybridization results involving decorated probes on microarrays can be predicted on the basis of thermodynamic data for duplex formation in solution. Predictable formation of isostable DNA not only benefits microarrays for gene expression analysis and genotyping, but may also improve the sequence-specificity of other applications that rely on the massively parallel formation of Watson-Crick duplexes.     

Selective pairing of polyfluorinated DNA bases

Novel selective non-hydrogen-bonding DNA base pairs utilizing fluorinated nucleoside analogues have been investigated. Melting studies of DNA duplexes containing 2,3,4,5-tetrafluorobenzene and 4,5,6,7-tetrafluoroindole bases on opposite strands show greater stabilization of the duplex compared with nonfluorinated hydrocarbon controls. Overall, these hydrophobic analogues are destabilizing compared with natural base pairs but are stabilizing compared with natural base mismatches. Such selective pairing may be due to solvent avoidance of these hydrophobic structures, burying their surfaces within the duplex. Our findings suggest that polyfluoroaromatic bases might be employed as a new, selective base-pairing system orthogonal to the natural genetic system.     

On the effect of covalently appended quinolones on termini of DNA duplexes

Quinolones are gyrase inhibitors that are widely used as antibiotics in the clinic. When covalently attached to oligonucleotides as 5'-acylamido substituents, quinolones were found to stabilize duplexes of oligonucleotides against thermal denaturation. For short duplexes, such as qu-T*GCGCA, where qu is a quinolone residue and T is a 5'-amino-5'-deoxythymidine residue, an increase in the UV melting point of up to 27.8 degrees C was measured. The stabilizing effect was demonstrated for all quinolones tested, namely nalidixic acid, oxolinic acid, pipemidic acid, cinoxacin, norfloxacin, and ofloxacin. The three-dimensional structure of (oa-T*GCGCA)2, where oa is an oxolinic acid residue, was solved by two-dimensional NMR spectroscopy and restrained molecular dynamics. In this complex, the oxolinic acid residues disrupt the terminal T1:A6 base pairs and stack on the G2:C5 base pairs. The displaced adenosine residues bind in the minor groove of the core duplex, while the thymidine residues pack against the oxolinic acid residues. The "molecular cap" thus formed fits tightly on the G:C base pairs, resulting in increased base-pairing fidelity, as demonstrated in UV melting experiments with the sequence oa-T*GGTTGAC and target strands containing a mismatched nucleobase. The structure of the "molecular cap" with its disrupted terminal base pair may also be helpful for modeling how quinolones block re-ligation of DNA strands in the active site of gyrases.     

Influence of metal coordination on the mismatch tolerance of ligand-modified PNA duplexes

Recent studies on metal incorporation in ligand-modified nucleic acids have focused on the effect of metal coordination on the stability of metal-containing duplexes or triplexes and on the metal binding selectivity but did not address the effect of the sequence of the nucleic acid in which the ligands are incorporated. We have introduced 8-hydroxyquinoline Q in 10-mer PNA strands with various sequences and have investigated the properties of the duplexes formed from these strands upon binding of Cu(2+). Variable-temperature UV-vis spectroscopy shows that, in the presence of Cu(2+), duplexes are formed even from ligand-modified Q-PNA strands that have a large number of mismatches. Spectrophotometric titrations demonstrate that at any temperature, one Cu(2+) ion binds a pair of Q-PNA strands that each contain one 8-hydroxyquinoline, but below the melting temperature, the PNA duplex exerts a supramolecular chelate effect, which prevents the transformation in the presence of excess Cu(2+) of the 1:2 Cu(2+):Q-PNA complexes into 1:1 complexes. EPR spectroscopy gives further support for the existence in the duplexes of [CuQ(2)] moieties that are similar to the corresponding square planar synthetic complex formed between Cu(2+) and 8-hydroxyquinoline. As PNA duplexes show a preferred handedness due to the chiral induction effect of a C-terminal l-lysine, which is transmitted through stacking interactions within the duplex, only if the metal-containing duplex has complementary strands, does it show a chiral excess measured by CD spectroscopy. The strong effect of the metal-ligand moiety is suggestive of an increased correlation length in PNA duplexes that contain such moieties. These results indicate that strong metal-ligand alternative base pairs significantly diminish the importance of Watson-Crick base pairing for the formation of a stable PNA duplex and lead to high mismatch tolerance, a principle that can be used in the construction of hybrid inorganic-nucleic acid nanostructures.     

Intrastrand locks increase duplex stability and base pairing selectivity

Oligodeoxynucleotide probes with disulfide locks between neighboring nucleobases show increases in melting point for duplexes with RNA target strands of up to 7.6 °C. The weakly pairing TT dimers are replaced with locked 2'-deoxy-5-(thioalkynyl)uridine residues via automated synthesis.     

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.     

Synthesis and pairing properties of alpha-tricyclo-DNA

We describe the synthesis of the phosphoramidite building blocks of alpha-tricyclo-DNA (alpha-tc-DNA) covering all four natural bases, starting from the already known corresponding alpha-tc-nucleosides. These building blocks were used for the preparation of three alpha-tc-oligonucleotide 10-mers representing a homopurine, a homopyrimidine, and a mixed purine/pyrimidine base sequence. The base-pairing properties with complementary parallel and antiparallel oriented DNA and RNA were studied by UV-melting analysis and CD spectroscopy. We found that alpha-tc-DNA binds preferentially to parallel nucleic acid complements through Watson-Crick duplex formation, with a preference for RNA over DNA. In comparison with natural DNA, alpha-tc-DNA shows equal to enhanced affinity to RNA and also pairs to antiparallel DNA or RNA complements, although with much lower affinity. In the mixed-base sequence these antiparallel duplexes are of the reversed Watson-Crick type, while in the homopurine/homopyrimidine sequences Hoogsteen and/or reversed Hoogsteen pairing is observed. Antiparallel duplex formation of two alpha-tc-oligonucleotides was also observed, although the thermal stability of this duplex was surprisingly low. The base-pairing properties of alpha-tc-DNA are discussed in the context of alpha-DNA, alpha-RNA, and alpha-LNA.     

NMR assessment of the global shape of a non-labelled DNA dodecamer containing a tandem of G-T mismatches

We have carried out a solution study of two non-labelled self-complementary DNA dodecamers d(GACTGTACAGTC)2 and d(GACTGTGCAGTC)2 by NMR, the second sequence composed of two G-T mismatches. Structures were determined using distances extracted from NOE effects alone or using both NOE and RDC constraints, measured in three different liquid crystalline media. We ensured that our data on the influence of the mesogen on the DNA structures, and the way in which the RDCs were incorporated as constraints in the protocol refinement, were consistent. We also tested the influence of different sets of RDCs and the best means of optimizing the calculation of D(a) and R. Resolution and accuracy of the ten best energy final structures were compared. The addition of a small set of RDC constraints significantly improves the final determined structures. We took advantage of the specificity of the RDC, i.e. it contains orientational information, and explored the global shape of the DNA duplexes; it was found that the duplexes do not have a large curvature. For the G-T base pair, we observed, in this particular sequence (tandem of G-T mismatches), a new pattern of base pairing, which involved the formation of a bifurcated hydrogen bond.     

Oligonucleotides with 1,4-dioxane-based nucleotide monomers

An epimeric mixture of H-phosphonates 5R and 5S has been synthesized in three steps from known secouridine 1. Separation of the epimers has been accomplished by RP-HPLC, allowing full characterization and incorporation of monomers X and Y into 9-mer oligonucleotides using H-phosphonates building blocks 5R and 5S, respectively. A single incorporation of either monomer X or monomer Y in the central position of a DNA 9-mer results in decreased thermal affinity toward both DNA and RNA complements (ΔT(m) = -3.5 °C/-3.5 °C for monomer X and ΔT(m) = -11.0 °C/-6.5 °C for monomer Y). CD measurements do not reveal major rearrangements of the duplexes formed, but molecular modeling suggests that local rearrangement of the sugar phosphate backbone and decreased base interactions with neighboring bases might be the origin of the decreased stability of duplexes.     

5'-Tethered stilbene derivatives as fidelity- and affinity-enhancing modulators of DNA duplex stability

A series of 5'-linked stilbene-DNA conjugates with different substituents in the distal aromatic ring of the stilbene was prepared, and the effect of the modifications on duplex stability was determined via UV-melting curves. A trimethoxystilbene derivative as a 5'-substituent increases duplex melting points by up to 12.2 degrees C per modification. With this alkoxystilbene substituent, terminal mismatches in DNA duplexes lower the melting point by up to 23.4 degrees C over the perfectly matched control, whereas terminal mismatches in unmodified DNA cause melting point depressions of no more than 6.1 degrees C. An aminomethylstilbene substituent linked to an oligopyrrolamide minor groove binder increases the melting point of an all-A/T decamer by up to 32.7 degrees C, thus shifting the melting point into a range typical for duplexes with statistical G/C-content. An affinity- and selectivity-enhancing effect was also observed when the trimethoxystilbene cap was employed on a small DNA microarray. The phosphoramidite of the trimethoxystilbene can be readily employed in automatic DNA synthesis, facilitating the generation of DNA chips with improved fidelity.     

Heterochiral DNA with Complementary Strands with α-d and β-d Configurations: Hydrogen-Bonded and Silver-Mediated Base Pairs with Impact of 7-Deazapurines Replacing Purines

Heterochiral DNA with hydrogen-bonded and silver-mediated base pairs have been constructed using complementary strands with nucleosides with α-d or β-d configuration. Anomeric phosphoramidites were employed to assemble the oligonucleotides. According to the T_m values and thermodynamic data, the duplex stability of the heterochiral duplexes was similar to that of homochiral DNA, but mismatch discrimination was better in heterochiral DNA. Replacement of purines by 7-deazapurines resulted in stable parallel duplexes, thereby confirming Watson–Crick-type base pairing. When cytosine was facing cytosine, thymine or adenine residues, duplex DNA formed silver-mediated base pairs in the presence of silver ions. Although the CD spectra of single strands with α-d configuration display mirror-like shapes to those with the β-d configuration, the CD spectra of the hydrogen-bonded duplexes and those with a limited number of silver pairs show a B-type double helix almost indistinguishable from natural DNA. Nonmelting silver ion–DNA complexes with entirely different CD spectra were generated when the number of silver ions was equal to the number of base pairs.     

B-DNA Structure and Stability: The Role of Nucleotide Composition and Order

We have quantum chemically analyzed the influence of nucleotide composition and sequence (that is, order) on the stability of double-stranded B-DNA triplets in aqueous solution. To this end, we have investigated the structure and bonding of all 32 possible DNA duplexes with Watson–Crick base pairing, using dispersion-corrected DFT at the BLYP-D3(BJ)/TZ2P level and COSMO for simulating aqueous solvation. We find enhanced stabilities for duplexes possessing a higher GC base pair content. Our activation strain analyses unexpectedly identify the loss of stacking interactions within individual strands as a destabilizing factor in the duplex formation, in addition to the better-known effects of partial desolvation. Furthermore, we show that the sequence-dependent differences in the interaction energy for duplexes of the same overall base pair composition result from the so-called “diagonal interactions” or “cross terms”. Whether cross terms are stabilizing or destabilizing depends on the nature of the electrostatic interaction between polar functional groups in the pertinent nucleobases.     

Synthesis and Properties of Isobicyclo‐DNA

We present the synthesis of the isobicyclo‐DNA building blocks with the nucleobases A, C, G and T, as well as biophysical and biological properties of oligonucleotides derived thereof. The synthesis of the sugar part was achieved in 5 steps starting from a known intermediate of the tricyclo‐DNA synthesis. Dodecamers containing single isobicyclo‐thymidine incorporations, fully modified A‐ and T‐containing sequences, and fully modified oligonucleotides containing all four bases were synthesized and characterized. Isobicyclo‐DNA forms stable duplexes with natural nucleic acids with a pronounced preference for DNA over RNA as complements. The most stable duplexes, however, arise by self‐pairing. Isobicyclo‐DNA forms preferentially B‐DNA‐like duplexes with DNA and A‐like duplexes with complementary RNA as determined by circular dichroism (CD) spectroscopy. Self‐paired duplexes show a yet unknown structure, as judged from CD spectroscopy. Biochemical tests revealed that isobicyclo‐DNA is stable in fetal bovine serum and does not elicit RNaseH activity.     

7-Deazapurine and 8-aza-7-deazapurine nucleoside and oligonucleotide pyrene "click" conjugates: synthesis, nucleobase controlled fluorescence quenching, and duplex stability

7-Deazapurine and 8-aza-7-deazapurine nucleosides related to dA and dG bearing 7-octadiynyl or 7-tripropargylamine side chains as well as corresponding oligonucleotides were synthesized. "Click" conjugation with 1-azidomethyl pyrene (10) resulted in fluorescent derivatives. Octadiynyl conjugates show only monomer fluorescence, while the proximal alignment of pyrene residues in the tripropargylamine derivatives causes excimer emission. 8-Aza-7-deazapurine pyrene "click" conjugates exhibit fluorescence emission much higher than that of 7-deazapurine derivatives. They are quenched by intramolecular charge transfer between the nucleobase and the dye. Oligonucleotide single strands decorated with two "double clicked" pyrenes show weak or no excimer fluorescence. However, when duplexes carry proximal pyrenes in complementary strands, strong excimer fluorescence is observed. A single replacement of a canonical nucleoside by a pyrene conjugate stabilizes the duplex substantially, most likely by stacking interactions: 6-12 °C for duplexes with a modified "adenine" base and 2-6 °C for a modified "guanine" base. The favorable photophysical properties of 8-aza-7-deazapurine pyrene conjugates improve the utility of pyrene fluorescence reporters in oligonucleotide sensing as these nucleoside conjugates are not affected by nucleobase induced quenching.     

Structural studies of LNA:RNA duplexes by NMR: conformations and implications for RNase H activity

We have used NMR and CD spectroscopy to study the conformations of modified oligonucleotides (locked nucleic acid, LNA) containing a conformationally restricted nucleotide (T(L)) with a 2'-O,4'-C-methylene bridge. We have investigated two LNA:RNA duplexes, d(CTGAT(L)ATGC):r(GCAUAUCAG) and d(CT(L)GAT(L)AT(L)GC):r(GCAUAUCAG), along with the unmodified DNA:RNA reference duplex. Increases in the melting temperatures of +9.6 degrees C and +8.1 degrees C per modification relative to the unmodified duplex were observed for these two LNA:RNA sequences. The three duplexes all adopt right-handed helix conformations and form normal Watson-Crick base pairs with all the bases in the anti conformation. Sugar conformations were determined from measurements of scalar coupling constants in the sugar rings and distance information derived from 1H-1H NOE measurements; all the sugars in the RNA strands of the three duplexes adopt an N-type conformation (A-type structure), whereas the sugars in the DNA strands change from an equilibrium between S- and N-type conformations in the unmodified duplex towards more of the N-type conformation when modified nucleotides are introduced. The presence of three modified T(L) nucleotides induces drastic conformational shifts of the remaining unmodified nucleotides of the DNA strand, changing all the sugar conformations except those of the terminal sugars to the N type. The CD spectra of the three duplexes confirm the structural changes described above. On the basis of the results reported herein, we suggest that the observed conformational changes can be used to tune LNA:RNA duplexes into substrates for RNase H: Partly modified LNA:RNA duplexes may adopt a duplex structure between the standard A and B types, thereby making the RNA strand amenable to RNase H-mediated degradation.     

Tuning the reaction site for enzyme-free primer-extension reactions through small molecule substituents

The replication of genetic information relies on the template-directed extension of DNA primers catalyzed by polymerases. The active sites of polymerases accept four different substrates and ensure fidelity and processivity for each of them. Because of the pivotal role of catalyzed primer extension for life, it is important to better understand this reaction on a molecular level. Here we present results from primer-extension reactions performed with chemical systems that show high reactivity in the absence of polymerases. Small molecular caps linked to the 5'-terminus of templates are shown to enhance the rate and selectivity of primer extension driven by 2-methylimidazolides as activated monomers for any of the four different templating bases (A, C, G, and T). The most consistent effect is provided by a stilbene carboxamide residue, rather than larger aromatic or aliphatic substituents. Up to 20-fold rate enhancements were achieved for the reactions at the terminus of the template. The preference for a medium size cap can be explained by competing interactions with both the oligonucleotides and the incoming deoxynucleotide. The data also show that there is no particularly intractable problem in combining promiscuity with fidelity. Exploratory experiments involving a longer template and a downstream-binding strand with a 5'-cap show up to 38-fold rate acceleration over the same reaction templated by a single overhanging nucleotide.     

Activation energies for dissociation of double strand oligonucleotide anions: evidence for watson-crick base pairing in vacuo

The dissociation kinetics of a series of complementary and noncomplementary DNA duplexes, (TGCA)(2) (3-), (CCGG)(2) (3-), (AATTAAT)(2) (3-), (CCGGCCG)(2) (3-), A(7).T(7) (3-), A(7).A(7) (3-), T(7).T(7) (3-), and A(7).C(7) (3-) were investigated using blackbody infrared radiative dissociation in a Fourier transform mass spectrometer. From the temperature dependence of the unimolecular dissociation rate constants, Arrhenius activation parameters in the zero-pressure limit are obtained. Activation energies range from 1.2 to 1.7 eV, and preexponential factors range from 10(13) to 10(19) s(-1). Dissociation of the duplexes results in cleavage of the noncovalent bonds and/or cleavage of covalent bonds leading to loss of a neutral nucleobase followed by backbone cleavage producing sequence-specific (a - base) and w ions. Four pieces of evidence are presented which indicate that Watson-Crick (WC) base pairing is preserved in complementary DNA duplexes in the gas phase: i. the activation energy for dissociation of the complementary dimer, A(7).T(7) (3-), to the single strands is significantly higher than that for the related noncomplementary A(7).A(7) (3-) and T(7).T(7) (3-) dimers, indicating a stronger interaction between strands with a specific base sequence, ii. extensive loss of neutral adenine occurs for A(7).A(7) (3-) and A(7).C(7) (3-) but not for A(7).T(7) (3-) consistent with this process being shut down by WC hydrogen bonding, iii. a correlation is observed between the measured activation energy for dissociation to single strands and the dimerization enthalpy (-DeltaH(d)) in solution, and iv. molecular dynamics carried out at 300 and 400 K indicate that WC base pairing is preserved for A(7).T(7) (3-) duplex, although the helical structure is essentially lost. In combination, these results provide strong evidence that WC base pairing can exist in the complete absence of solvent.     

Aminoglycoside complexation with a DNA.RNA hybrid duplex: the thermodynamics of recognition and inhibition of RNA processing enzymes

Spectroscopic and calorimetric techniques were employed to characterize and contrast the binding of the aminoglycoside paromomycin to three octamer nucleic acid duplexes of identical sequence but different strand composition (a DNA.RNA hybrid duplex and the corresponding DNA.DNA and RNA.RNA duplexes). In addition, the impact of paromomycin binding on both RNase H- and RNase A-mediated cleavage of the RNA strand in the DNA.RNA duplex was also determined. Our results reveal the following significant features: (i) Paromomycin binding enhances the thermal stabilities of the RNA.RNA and DNA.RNA duplexes to similar extents, with this thermal enhancement being substantially greater in magnitude than that of the DNA.DNA duplex. (ii) Paromomycin binding to the DNA.RNA hybrid duplex induces CD changes consistent with a shift from an A-like to a more canonical A-conformation. (iii) Paromomycin binding to all three octamer duplexes is linked to the uptake of a similar number of protons, with the magnitude of this number being dependent on pH. (iv) The affinity of paromomycin for the three host duplexes follows the hierarchy, RNA.RNA > DNA.RNA > DNA.DNA. (v) The observed affinity of paromomycin for the RNA.RNA and DNA.RNA duplexes decreases with increasing pH. (vi) The binding of paromomycin to the DNA.RNA hybrid duplex inhibits both RNase H- and RNase A-mediated cleavage of the RNA strand. We discuss the implications of our combined results with regard to the specific targeting of DNA.RNA hybrid duplex domains and potential antiretroviral applications.     

Base pairing and replicative processing of the formamidopyrimidine-dG DNA lesion

The 2,6-diamino-4-hydroxy-5-formamidopyrimidine of 2'-deoxyguanosine (FaPydG) is one of the major DNA lesions found after oxidative stress in cells. To clarify the base pairing and coding potential of this major DNA lesion with the aim to estimate its mutagenic effect, we prepared oligonucleotides containing a cyclopentane based analogue of the DNA lesion (cFaPydG). In addition, oligonucleotides containing the cyclopentane analogue of 2'-deoxyguanosine (cdG), and oligonucleotides containing 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) were synthesized. The thermodynamic stability of duplexes containing these building blocks and all canonical counterbases were determined by concentration dependent melting-point measurements (van't Hoff plots). The data reveal that cFaPydG greatly destabilizes a DNA duplex (DeltaDeltaG degrees (298K) approximately 2-4 kcal mol(-1)). The optimal base pairing partner for the cFaPydG lesion is dC. Investigation of duplexes containing dG and cdG shows that the effect of substituting the deoxyribose by a cyclopentane moiety is marginal. The data also provide strong evidence that the FaPydG lesion is unable to form a base pair with dA. Our computational studies indicate that the syn-conformation required for base pairing with dA is energetically unfavorable. This is in contrast to 8-oxodG for which the syn-conformation represents the energetic minimum. Kinetic primer extension studies using S. cerevisiae Pol eta reveal that cFaPydG is replicated in an error-free fashion. dC is inserted 2-3 orders of magnitude more efficiently than dT or dA, showing that FaPydG is a lesion which retains the coding potential of dG. This is also in contrast to 8-oxodG, for which base pairing with dC and dA was established.