Influence of base dynamics on the conformational properties of DNA: Observation of static conformational states in rigid duplexes at 77 K

Time-resolved fluorescence of 2-aminopurine-labeled DNA duplexes at 77 K reveals the relationship between base dynamics and the conformational heterogeneity that results in the well-known multiexponential fluorescence decay at room temperature. The conformation that exhibits rapid interbase charge transfer at room temperature is not populated in the frozen duplex at 77 K; this geometry is accessed by thermal motion of the bases, it is not a minimum energy structure of the duplex. Three photophysically distinct conformational states persist in the frozen duplex; these are minimum energy structures and do not interconvert at room temperature on the time scale of the 2-aminopurine excited-state lifetime.     

Energetics of nucleic acid stability: the effect of DeltaCP

We report high-resolution differential scanning calorimetric data on the poly(dAdT)poly(dAdT), poly(dA)poly(dT), poly(dIdC)poly(dIdC), poly(dGdC)poly(dGdC), poly(rA)poly(rU), and poly(rI)poly(rC) nucleic acid duplexes. We use these data to evaluate the melting temperatures, TM, enthalpy changes, DeltaHM, and heat capacity changes, DeltaCP, accompanying helix-to-coil transitions of each polymeric duplex studied in this work at different NaCl concentrations. In agreement with previous reports, we have found that DeltaCP exhibits a positive, nonzero value, which, on average, equals 268 +/- 33 J mol(-1) K(-1). With DeltaCP, we have calculated the transition free energies, DeltaG, enthalpies, DeltaH, and entropies, DeltaS, for the duplexes as a function of temperature. Since, DeltaG, DeltaH, and DeltaS all strongly depend on temperature, the thermodynamic comparison between DNA and/or RNA duplexes (that may differ from one another with respect to sequence, composition, conformation, etc.) is physically meaningful only if extrapolated to a common temperature. We have performed such comparative analyses to derive differential thermodynamic parameters of formation of GC versus AT, AU, and IC base pairs as well as B' versus A and B helix conformations. We have proposed some general microscopic interpretations for the observed sequence-specific and conformation-specific thermodynamic differences between the duplexes.     

Photosensitizing properties of biopterin and its photoproducts using 2'-deoxyguanosine 5'-monophosphate as an oxidizable target

UV-A radiation (320-400 nm) induces damage to the DNA molecule and its components through photosensitized reactions. Biopterin (Bip) and its photoproducts 6-formylpterin (Fop) and 6-carboxypterin (Cap) accumulate in the skin of human beings suffering from vitiligo, a depigmentation disorder where the protection against UV radiation fails because of the lack of melanin. This study was aimed to evaluate the photosensitizing properties of oxidized pterins present in the skin and to elucidate the mechanisms involved in the photosensitized oxidation of purine nucleotides by pterins in vitro. For this purpose, steady-state and time-resolved experiments in acidic (pH 5.0-5.8) aqueous solution were performed using Bip, Fop and Cap as photosensitizers and the nucleotide 2'-deoxyguanosine 5'-monophosphate (dGMP) as an oxidizable target. The three pterin derivatives are able to photosensitize dGMP, being Fop the most efficient sensitizer. The reactions proceed through two competing pathways: (1) electron transfer from dGMP to triplet excited-state of pterins (type I mechanism) and (2) reaction of dGMP with (1)O(2) produced by pterins (type II mechanism). Kinetic analysis revealed that the electron transfer pathway is the main mechanism and the interaction of dGMP with the triplet excited-state of pterins and the formation of the corresponding dGMP radicals were demonstrated by laser flash photolysis experiments. The biological implications of the results obtained are also discussed.     

Thermodynamics of interactions of water-soluble porphyrins with RNA duplexes

We characterized the interactions of meso-tetrakis(4N-(2-hydroxyethyl)pyridinium-4-yl) porphyrin (TEtOHPyP4), meso-tetrakis(4N-allylpyridinium-4-yl) porphyrin (TAlPyP4), and meso-tetrakis(4N-metallylpyridinium-4-yl) porphyrin (TMetAlPyP4) with the poly(rA)poly(rU) and poly(rI)poly(rC) RNA duplexes between 18 and 45 degrees C by employing circular dichroism, light absorption, and fluorescence intensity spectroscopic measurements. Our results suggest that TEtOHPyP4 and TAlPyP4 intercalate into the poly(rA)poly(rU) and poly(rI)poly(rC) host duplexes, while TMetAlPyP4 associates with these RNA duplexes by forming outside-bound, self-stacked aggregates. We used our temperature-dependent absorption titration data to determine the binding constants and stoichiometry for each porphyrin-RNA binding event studied in this work. From the temperature dependences of the binding constants, we calculated the binding free energies, DeltaG(b), enthalpies, DeltaH(b), and entropies, DeltaS(b). For each RNA duplex, the binding enthalpy, DeltaH(b), is the most favorable for TEtOHPyP4 (an intercalator) followed by TAlPyP4 (an intercalator) and TMetAlPyP4 (an outside binder). On the other hand, for each duplex, external self-stacking of TMetAlPyP4 produces the most favorable change in entropy, DeltaS(b), followed by the intercalators TAlPyP4 and TEtOHPyP4. Thus, our results suggest that the thermodynamic profile of porphyrin-RNA binding may correlate with the binding mode. This correlation reflects the differential nature of molecular forces that stabilize/destabilize the two modes of binding-intercalation versus external self-stacking along the host duplex.     

Hydration changes accompanying nucleic acid intercalation reactions:volumetric characterizations

We use high precision ultrasonic velocimetric and densimetric techniques to determine at 25 degrees C the changes in volume, deltaV, and adiabatic compressibility, deltaK(S), that accompany the binding of ethidium to the poly(rA)poly(rU), poly(dAdT)poly(dAdT), poly(dGdC)poly(dGdC), and poly(dIdC)poly(dIdC) duplexes, as well as to the poly(rU)poly(rA)poly(rU) triplex. The binding of ethidium to each of the duplexes and the triplex is accompanied by negative changes in volume, deltaV, and adiabatic compressibility, deltaK(S). We discuss the basis for relating macroscopic and microscopic properties, particularly, emphasizing how measured changes in volume and compressibility can be quantitatively interpreted in terms of the differential hydration properties of DNA and RNA structures in their ligand-free and ligand-bound states. We also estimate the entropic cost of intercalation-induced changes in hydration of each of the nucleic acid structures and the drug. In general, our results emphasize the vital role of hydration in modulating the energetics of drug-DNA binding, while also underscoring the fact that hydration must be carefully taken into account in analysis and prediction of the energetics of nucleic acid recognition.     

On the apparently anomalous distance dependence of charge-transfer rates in 9-amino-6-chloro-2-methoxyacridine-modified DNA

From previous thermal and photoinduced charge-transfer reactions in duplex DNA there is accumulative evidence for an attenuation parameter beta of the distance dependence in the range 0.6-0.8 A(-1), with the exception of one specific system exhibiting beta = 1.5 A(-1) which is reinvestigated in this paper. Femtosecond to nanosecond time-resolved pump-probe spectroscopy has been used to follow photoinduced charge-shift dynamics in DNA duplexes containing a covalently appended, protonated 9-alkylamino-6-chloro-2-methoxyacridine chromophore. This acridine derivative (X+) resides in the DNA duplex at a specific abasic site, which is highly defined as reflected in the monoexponentiality of the kinetics. In the presence of only neighboring A:T base pairs, no charge transfer occurs within the excited-state lifetime (18 ns) of the chromophore. However, the presence of a guanine nucleobase as either a nearest neighbor or with one interspersed A:T base pair does result in fluorescence quenching. In the case of nearest neighbors, the intermediate radical state X* is formed within 4 ps and decays on the 30 ps time scale. Placing one A:T base pair between the X+ and guanine slows down the forward transfer rate by 3 orders of magnitude, corresponding to an apparent beta value of >2.0 A(-1). This dramatic decrease in the rate is due to a change in charge-transfer mechanism from a (nearly) activationless to a thermally activated regime in which the forward transfer is slower than the back transfer and the X* state is no longer observed. These observations indicate that the distance dependence of charge injection in the X+-labeled DNA duplex is not solely caused by a decrease in electronic couplings but also by a concomitant increase of the activation energy with increasing distance. This increase in activation energy may result from the loss of driving force due to excited-state relaxation competing with charge transfer, or reflect distance-dependent changes in the energetics, predominantly of the low-frequency reorganization energy in this charge-shift reaction, on purely electrostatic grounds. To test the hypothesis of distance-dependent activation energy, guanine has been replaced by 7-deazaguanine, its easier-to-oxidize purine analogue. In these duplexes, a similar change of charge-transfer mechanism is found. However, consistent with an a priori larger driving force this change occurs at a larger donor-acceptor separation than in the X+-guanine systems. Independent of the detailed contributions to the distance-dependent activation energy, this phenomenon illustrates the complex nature of experimental beta values.     

Dissociation energies of deoxyribose nucleotide dimer anions measured using blackbody infrared radiative dissociation

The dissociation kinetics of deprotonated deoxyribose nucleotide dimers were measured using blackbody infrared radiative dissociation. Experiments were performed with noncovalently bound dimers of phosphate, adenosine (dAMP), cytosine (dCMP), guanosine (dGMP), thymidine (dTMP), and the mixed dimers dAMP.dTMP and dGMP.dCMP. The nucleotide dimers fragment through two parallel pathways, resulting in formation of the individual nucleotide or nucleotide + HPO3 ion. Master equation modeling of this kinetic data was used to determine threshold dissociation energies. The dissociation energy of (dGMP.dCMP-H)- is much higher than that for the other nucleotide dimers. This indicates that there is a strong interaction between the nucleobases in this dimer, consistent with the existence of Watson-Crick hydrogen bonding between the base pairs. Molecular mechanics simulations indicate that Watson-Crick hydrogen bonding occurs in the lowest energy structures of (dGMP.dCMP-H)-, but not in (dAMP.dTMP-H)-. The trend in gas phase dissociation energies is similar to the trend in binding energies measured in nonaqueous solutions within experimental error. Finally, the acidity ordering of the nucleotides is determined to be dTMP < dGMP < dCMP < dAMP, where dAMP has the highest acidity (largest delta Gacid).     

EXCITED-STATE PROPERTIES OF THE ALTERNATING POLYNUCLEOTIDE POLY(dA-dT)POLY(dA-dT)

Measurements of the steady-state fluorescence spectrum and anisotropy, r, of the alternating polynucleotide poly(dA-dT).poly(dA-dT) were carried out in order to characterize its photophysical properties at room temperature. The shape of the fluorescence spectrum depends on the excitation wavelength, namely, the relative fluorescence intensity of the short-wavelength peak decreases for excitation at short wavelengths. When monitoring the emission at short wavelengths, r is 0.18 and independent of the excitation wavelength. When monitoring the emission at long wavelengths, however, r is very low, about 0.03. These results suggest that: (i) the short-wavelength emission stems from thymine; and (ii) the long-wavelength emission stems from an excited-state complex (excimer), with the same one being formed regardless of whether thymine or adenine is excited. The corresponding fluorescence spectra have been resolved. The occurrence of transfer of electronic energy is discussed.     

On the stability of double stranded nucleic acids

We present the first pressure-versus-temperature phase diagram for the helix-to-coil transition of double stranded nucleic acids. The thermodynamic stability of a nucleic acid duplex is a complex function of temperature and pressure and strongly depends on the denaturation temperature, T(M), of the duplex at atmospheric pressure. Depending upon T(M), pressure, and temperature, the phase diagram shows that pressure may stabilize, destabilize, or have no effect on the conformational state of DNA. To verify the phase diagram, we have conducted high-pressure UV melting experiments on poly(dIdC)poly(dIdC), a DNA duplex, poly(rA)poly(rU), an RNA duplex, and poly(dA)poly(rU), a DNA/RNA hybrid duplex. The T(M) values of these duplexes have been modulated by altering the solution ionic strength. Significantly, at low salt, these three duplexes have helix-to-coil transition temperatures of 50 degrees C or less. In agreement with the derived phase diagram, we found that the polymeric duplexes were destabilized by pressure if the T(M) is < approximately 50 degrees C. However, these duplexes were stabilized by pressure if the T(M) is > approximately 50 degrees C. The DNA/RNA hybrid duplex, poly(dA)poly(rU), with a T(M) of 31 degrees C in 20 mM NaCl undergoes a pressure-induced helix-to-coil transition at room temperature. This is the first report of pressure-induced denaturation of a nucleic acid duplex and provides new insights into the molecular forces stabilizing these structures.     

Fluorescence of the DNA double helix (dA)20 x (dT)20 studied by femtosecond spectroscopy--effect of the duplex size on the properties of the excited states

The fluorescence of the DNA double-stranded oligomer (dA)20 x (dT)20 is studied at room temperature by fluorescence up-conversion at times shorter than 10 ps. The profile of the up-conversion spectra is similar to that of the steady-state fluorescence spectrum, showing that the majority of the photons are emitted within the probed time scale. At all the probed wavelengths, the fluorescence decays are slower than those of the monomeric chromophores dAMP and TMP. The fluorescence anisotropy decays show strong wavelength dependence. These data allow us to conclude that energy transfer takes place in this double helix and that this process involves exciton states. The spectral and dynamical properties of the oligomer are compared to those of the polymer poly(dA) x poly(dT), composed of about 2000 base pairs, reported previously. The oligomer absorption spectrum is characterized by a smaller hypsochromic shift and weaker hypochromism compared to the polymer. Moreover, the fluorescence decays of (dA)20 x (dT)20 are twice as fast as those of poly(dA) x poly(dT), and its fluorescence anisotropy decays more slowly. These differences are the fingerprints of a larger delocalization of the excited states induced by an increase in the size of the duplex.     

Donor/acceptor interactions in systematically modified Ru(II)-Os(II) oligonucleotides

Donor/acceptor (D/A) interactions are studied in a series of doubly modified 19-mer DNA duplexes. An ethynyl-linked Ru(II) donor nucleoside is maintained at the 5' terminus of each duplex, while an ethynyl-linked Os(II) nucleoside, placed on the complementary strands, is systematically moved toward the other terminus in three base pair increments. The steady-state Ru(II)-based luminescence quenching decreases from 90% at the shortest separation of 16 A (3 base pairs) to approximately 11% at the largest separation of 61 A (18 base pairs). Time-resolved experiments show a similar trend for the Ru(II) excited-state lifetime, and the decrease in the averaged excited-state lifetime for each duplex is linearly correlated with the fraction quenched obtained by steady-state measurements. Analysis according to the F?rster dipole-dipole energy transfer mechanism shows a reasonable agreement. Deviation from idealized behavior is primarily attributed to uncertainty in the orientation factor, kappa(2). Analyzing D/A interactions in an analogous series of doubly modified oligonucleotides, where the ethynyl-linked Ru(II) center is replaced with a saturated two-carbon linked complex, yields an excellent correlation with the F?rster mechanism. As this simple change partially relaxes the rigid geometry of the donor chromophore, these results suggest that the deviation from idealized F?rster behavior observed for the duplexes containing the rigidly held Ru(II) center originates, at least partially, from ambiguities in the orientation factor. Surprisingly, analyzing both quenching data sets according to the Dexter mechanism also shows an excellent correlation. Although this can be interpreted as strong evidence for a Dexter triplet energy transfer mechanism, it does not imply that this electron exchange mechanism is operative in these D/A duplexes. Rather, it suggests that systems that transfer energy via the F?rster mechanism can under certain circumstances exhibit Dexter-like "behavior", thus illustrating the danger of imposing a single physical model to describe D/A interactions in such complex systems. While we conclude that the F?rster dipole-dipole energy transfer mechanism is the dominant pathway for D/A interactions in these modified oligonucleotides, a minor contribution from the Dexter electron exchange mechanism at short distances is likely. This complex behavior distinguishes DNA-bridged Ru(II)/Os(II) dyads from their corresponding low molecular-weight and covalently attached counterparts.     

Recognition properties of donor- and acceptor-modified biphenyl-DNA

The recognition properties of DNA duplexes containing single or triple incorporations of eight different donor-modified (OMe, NH(2)) and acceptor-modified (NO(2)) biphenyl residues as base replacements in opposite positions were probed by UV-melting and by CD and fluorescence spectroscopy. We found a remarkable dependence of duplex stability on the natures of the substituents (donor vs. acceptor). The stabilities of duplexes with one biphenyl pair increase in the order donor/donor < acceptor/donor < acceptor/acceptor substitution. The most stable biphenyl pairs stabilize duplexes by up to 6 degrees C in T(m). In duplexes with three consecutive biphenyl pairs the stability increases in the inverse order (acceptor/acceptor < donor/acceptor < donor/donor) with increases in T(m), relative to an unmodified duplex, of up to 10 degrees C. A thermodynamic analysis, combined with theoretical calculations of the physical properties of the biphenyl substituents, suggests that in duplexes with single biphenyl pairs the affinity is dominated by electrostatic forces between the biphenyl/nearest neighbor natural base pairs, whereas in the triple-modified duplexes the increase in thermal stability is predominantly determined by hydrophobic interactions of the biphenyl residues with each other. Oligonucleotides containing amino biphenyl residues are fluorescent. Their fluorescence is largely quenched when they are paired with themselves or with nitrobiphenyl-containing duplex partners.     

Stereospecific synthesis and characterization of oligodeoxyribonucleotides containing an N2-(1-carboxyethyl)-2'-deoxyguanosine

Methylglyoxal is a highly reactive alpha-ketoaldehyde that is produced endogenously and present in the environment and foods. It can modify DNA and proteins to form advanced glycation end products (AGEs). Emerging evidence has shown that N2-(1-carboxyethyl)-2'-deoxyguanosine (N2-CEdG) is a major marker for AGE-linked DNA adducts. Here, we report, for the first time, the preparation of oligodeoxyribonucleotides (ODNs) containing individual diastereomers of N2-CEdG via a postoligomerization synthesis method, which provided authentic substrates for examining the replication and repair of this lesion. In addition, thermodynamic parameters derived from melting temperature data revealed that the two diastereomers of N2-CEdG destabilized significantly the double helix as represented by a 4 kcal/mol increase in Gibbs free energy for duplex formation at 25 degrees C. Primer extension assay results demonstrated that both diastereomers of N2-CEdG could block considerably the replication synthesis mediated by the exonuclease-free Klenow fragment of Escherichia coli DNA polymerase I. Strikingly, the polymerase incorporated incorrect nucleotides, dGMP and dAMP, opposite the lesion more preferentially than the correct nucleotide, dCMP.     

Directional Preference of DNA‐Mediated Electron Transfer in Gold‐Tethered DNA Duplexes: Is DNA a Molecular Rectifier?

Electrical properties of self‐assembling DNA nanostructures underlie the paradigm of nanoscale bioelectronics, and as such require clear understanding. DNA‐mediated electron transfer (ET) from a gold electrode to DNA‐bound Methylene Blue (MB) shows directional preference, and it is sequence‐specific. During the electrocatalytic reduction of [Fe(CN)₆]^3? catalyzed by DNA‐bound MB, the ET rate constant for DNA‐mediated reduction of MB reaches (1.32±0.2)10³ and (7.09±0.4)10³ s^?1 for (dGdC)₂₀ and (dAdT)₂₅ duplexes. The backward oxidation process is less efficient, making the DNA duplex a molecular rectifier. Lower rates of ET via (dGdC)₂₀ agree well with its disturbed π‐stacked sub‐molecular structure. Such direction‐ and sequence‐specific ET may be implicated in DNA oxidative damage and repair, and be relevant to other polarized surfaces, such as cell membranes and biomolecular interfaces.     

Interactions of mitoxantrone with duplex and triplex DNA studied by electrospray ionization mass spectrometry

We have examined interactions between mitoxantrone (MXT) and DNA duplexes or triplexes with different base compositions by using electrospray ionization mass spectrometry (ESI‐MS), respectively. MXT interacts preferentially with DNA duplexes compared to the triplexes. In the mass spectrum of the duplex–MXT mixture, the complex peaks dominated in the ratios of duplex/MXT of 1:1, 1:2 and 1:3, and the 1:2 duplex/MXT peak was the most abundant. In contrast, only 1:1 triplex–MXT complexes were observed in the mass spectrum of the triplex–MXT mixture, and the most intensive peak was a free triplex ion without MXT. Moreover, no sequence selectivity of MXT to different DNA duplexes was found while MXT showed greater affinity to the triplexes that have adjacent TAT or C⁺GC sequences. In the course of sustained off‐resonance irradiation collision‐induced dissociation (SORI‐CID), the MXT‐duplex complexes generated two separated strands, and the MXT remained on the purine strand side. UV/Vis spectra showed that MXT interacted with DNA by intercalation. Compared with emodin (a duplex intercalator) and napthylquinoline (a triplex binder), we found that the side chain of MXT might play a role in the binding of MXT to the duplexes and the triplexes. ESI‐MS shows an advantage in speed and straightforwardness for the study of drug interactions with nucleic acids. Copyright © 2008 John Wiley & Sons, Ltd.     

B-DNA Helix Stability in a Solvent-Free Environment

B-DNA is the most common DNA helix conformation under physiological conditions. However, when the amount of water in a DNA solution is decreased, B-to-A helix transitions have been observed. To understand what type of helix conformations exist in a solvent-free environment, a series of poly d(CG)(n) and mixed sequence DNA duplexes from 18 to 30 bp were examined with circular dichroism (CD), ESI-MS, ion mobility, and molecular dynamics. From the CD spectra, it was observed that all sequences had B-form helices in solution. However, the solvent-free results were more complex. For the poly d(CG)(n) series, the 18 bp duplex had an A-form helix conformation, both A- and B-helices were present for the 22 bp duplex, and only B-helices were observed for the 26 and 30 bp duplexes. Since these sequences were all present as B-DNA in solution, the observed solvent-free structures illustrate that smaller helices with fewer base pairs convert to A-DNA more easily than larger helices in the absence of solvent. A similar trend was observed for the mixed sequence duplexes where both an A- and B-helix were present for the 18 bp duplex, while only B-helices occur for the larger 22, 26, and 30 bp duplexes. Since the solvent-free B-helices appear at smaller sizes for the mixed sequences than for the pure d(CG)(n) duplexes, the pure d(CG)(n) duplexes have a greater A-philicity.     

Spectroscopic characterization of fluorescein- and tetramethylrhodamine-labeled oligonucleotides and their complexes with a DNA template

We measured absorption and emission spectra, fluorescence quantum yield, anisotropy, fluorescence resonance energy transfer (FRET), and melting temperature to characterize fluorescein- and tetramethylrhodamine (TMR)-labeled oligonucleotides in solution and when hybridized to a common DNA template. Upon hybridization to the template, both the absorption and emission spectra of TMR-labeled duplexes exhibited a shift with respect to those of labeled oligonucleotides, depending on the location of the TMR on the oligonucleotide. Measurements of quantum yield, anisotropy, and melting temperature indicated that TMR interacted with nucleotides within the duplexes in the order (T1>T5>T11, T16) that the oligonucleotide with TMR labeled at the 5' end (T1) is stronger than that labeled at position 5 from the 5' end (T5), which is also stronger than those labeled at the positions, 11 and 16, from the 5' end (T11, T16). In the case of the duplex formed between T1 and the template, fluorescence quenching was observed, which is attributed to the interaction between the dye molecule and guanosines located at the single-stranded portion of the template. A two-state model was suggested to describe the conformational states of TMR in the duplex. The melting temperatures of the four FRET complexes show the same pattern as those of TMR-labeled duplexes. We infer that the interactions between TMR and guanosine persist in the FRET complexes. This interaction may bring the donor and the acceptor molecules closely together, which could cause interaction between the two dye molecules shown in absorbance measurements of the FRET complexes.     

Observation of slow charge redistribution preceding excited-state proton transfer

The photoacid 8-hydroxy-N,N,N',N',N',N'-hexamethylpyrene-1,3,6-trisulfonamide (HPTA) and related compounds are used to investigate the steps involved in excited-state deprotonation in polar solvents using pump-probe spectroscopy and time correlated single photon counting fluorescence spectroscopy. The dynamics show a clear two-step process leading to excited-state proton transfer. The first step after electronic excitation is charge redistribution occurring on a tens of picoseconds time scale followed by proton transfer on a nanosecond time scale. The three states observed in the experiments (initial excited state, charge redistributed state, and proton transfer state) are recognized by distinct features in the time dependence of the pump-probe spectrum and fluorescence spectra. In the charge redistributed state, charge density has transferred from the hydroxyl oxygen to the pyrene ring, but the OH sigma bond is still intact. The experiments indicate that the charge redistribution step is controlled by a specific hydrogen bond donation from HPTA to the accepting base molecule. The second step is the full deprotonation of the photoacid. The full deprotonation is clearly marked by the growth of stimulated emission spectral band in the pump-probe spectrum that is identical to the fluorescence spectrum of the anion.     

Genotyping by alkaline dehybridization using graphically encoded particles

This work describes a nonenzymatic, isothermal genotyping method based on the kinetic differences exhibited in the dehybridization of perfectly matched (PM) and single-base mismatched (MM) DNA duplexes in an alkaline solution. Multifunctional encoded hydrogel particles incorporating allele-specific oligonucleotide (ASO) probes in two distinct regions were fabricated by using microfluidic-based stop-flow lithography. Each particle contained two distinct ASO probe sequences differing at a single base position, and thus each particle was capable of simultaneously probing two distinct target alleles. Fluorescently labeled target alleles were annealed to both probe regions of a particle, and the rate of duplex dehybridization was monitored by using fluorescence microscopy. Duplex dehybridization was achieved through an alkaline stimulus using either a pH step function or a temporal pH gradient. When a single target probe sequence was used, the rate of mismatch duplex dehybridization could be discriminated from the rate of perfect match duplex dehybridization. In a more demanding application in which two distinct probe sequences were used, we found that the rate profiles provided a means to discriminate probe dehybridizations from both of the two mismatched duplexes as well as to distinguish at high certainty the dehybridization of the two perfectly matched duplexes. These results demonstrate an ability of alkaline dehybridization to correctly discriminate the rank hierarchy of thermodynamic stability among four sets of perfect match and single-base mismatch duplexes. We further demonstrate that these rate profiles are strongly temperature dependent and illustrate how the sensitivity can be compensated beneficially by the use of an actuating gradient pH field.     

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.