3'-Ferrocene-labeled oligonucleotide chains end-tethered to gold electrode surfaces: novel model systems for exploring flexibility of short DNA using cyclic voltammetry

The flexibility of DNA is of central importance in biology, medicine, materials science, and mechanical engineering. In this study, we report an unprecedented electrochemical approach for investigating the flexibility of a short (typically 20-base), surface end-tethered single-stranded synthetic DNA oligonucleotide and of its postformed DNA duplex, taking as an example the homopolymer (dT)20 sequence in the regime of very high ionic strength ( approximately 1 M).     

Peculiarities of the DNA hybridization on the surface of bilayer lipid membranes

The method of electrostriction was applied to study the peculiarities of interaction of short oligonucleotides with free standing (BLM) and supported lipid membranes (sBLM) and of the duplex formation between complementary oligonucleotides on a membrane surface. The 15-mer single stranded DNA (pentadecathymidylate-(dT)(15)) was modified either with cholesterol (CH(dT)(15)) or by palmitoyl chain (C16(dT)(15)). The interaction of CH(dT)(15) with free standing BLM or with BLM formed on an agar or gold support was accompanied by sharp increase of elasticity modulus in direction perpendicular to the membrane plane ,E( perpendicular), and by increase of surface potential. In contrast, C16(dT)(15) did not induce substantial changes of elasticity modulus, however, the surface potential was changed in a similar manner as for CH(dT)(15). Hybridization of DNA following addition of complementary chain (dA)(15) has been accompanied by a small decrease of elasticity modulus and by a slight increase of surface potential. Both the incorporation of chemically modified oligonucleotides into the lipid bilayer as well as hybridization of DNA are not cooperative processes as has been demonstrated by analysis using Scatchard plot of corresponding values.     

Parallel-stranded DNA: enhancing duplex stability by the 'G-clamp' and a pyrrolo-dC derivative

The new pyrrolo-dC derivative 4 tethered with an alkylamino side chain via a triazole linker was synthesized. Oligonucleotides containing the G-clamp 3 or the pyrrolo-dC derivative 4 were prepared. Oligonucleotide synthesis and deprotection under standard conditions led to unwanted side product formation. The side product was identified as an acrylonitrile adduct of the aminoalkyl side chain. Changing the synthesis and work-up conditions to fast-deprotection chemistry and performing β-elimination of the cyanoethyl group on the solid support yielded pure oligonucleotides. Oligonucleotide duplexes with parallel chain orientation were constructed incorporating dA·dT and isoG(d)·dC base pairs. Replacement of dC-residues by the G-clamp 3 led to extraordinarily stable duplexes (ΔT(m) = +11 °C for two incorporations) in ps DNA, while the pyrrolo-dC derivative 4 behaved like dC. Surprisingly, the G-clamp 3 forms an even more stable base pair with 2'-deoxyisoguanosine in DNA with parallel chain orientation than with 2'-deoxyguanosine in aps DNA.     

Electron transport by molecular motion of redox-DNA strands: unexpectedly slow rotational dynamics of 20-mer ds-DNA chains end-grafted onto surfaces via C6 linkers

The dynamics of electron transport within molecular layers of 3'-ferrocenylated 20-mer oligonucleotide, 5'-thiol end-grafted onto gold electrode surfaces via a six-carbon (C6) linker, is studied by cyclic voltammetry. Single-stranded Fc-DNA layers are observed to behave as diffusionless systems reflecting the rapid dynamics of the ssDNA strand. Following hybridization, the Fc-dsDNA-C6 layers give rise to a characteristic cyclic voltammetry behavior evidencing that the Fc head is animated by a purely diffusional motion, which is ascribed to free rotation of the rigid DNA duplex around its C6 anchoring linker. A model, describing the motion of the Fc head as resulting from hinge motion of the DNA duplex, is developed allowing the motional dynamics of the Fc-dsDNA-C6 chains to be quantified in terms of an apparent rotational diffusion coefficient, Dr. The value found for Dr is approximately 3-4 orders of magnitude slower than expected for free rotation of dsDNA in solution, pointing to a drastic motion-slowing role of the anchoring surface. Accessibility of the Fc head for the electron transfer at the electrode is also shown to modulate the apparent dsDNA dynamics. The dynamics of Fc-dsDNA-C 6 is found to be insensitive to the presence of a single mismatch in the middle of the strand, confirming that charge transport by dsDNA conduction (DNA CT) is not present for the systems studied here. However, electron transport by free hinge motion of the dsDNA chain is shown to be fast enough to, a priori, compete favorably with DNA CT.     

Response of DNA fragments to potentiometric sensors studied using HPLC

Potentiometric sensors are studied as viable candidates for the construction of high throughput DNA arrays. For preliminary investigations, such sensors were used in an HPLC setup in the present work. This avoided errors due to ionic contaminants or additives in the commercial samples. The oligonucleotides dT(10), dT(20) and dT(30) were used as test substances. The potentiometric sensors were of the coated wire type, containing PVC, DOP, MTDDACl and a synthetic podand urea receptor. The HPLC system consisted of a reversed phase column eluted with a phosphate buffer, triethylammoniumacetate (TEAA), and an acetonitrile gradient. Molar responses and sensitivities increased with increasing chain length of oligonucleotides, yielding detection limits as low as 10(-6)M (dT(30), injected concentration). The slopes of the calibration graphs were at least 23 mV/decade (dT(10)), which was much higher than expected. The results are discussed in view of the potential use of this sensor type in high throughput microarrays.     

Construction of a DNA-Based Supramolecular Nanosheet That Emits Bluish-White Light from Charge-Transfer Excited States of the Nucleobases

1,ω-Inosinic acid-bearing bolaamphiphiles dI(18), dI(19), and dI(20) with a 3′-phosphorylated inosine as a universal base connected to each end of an oligomethylene chain were synthesized for the first time. Single-component self-assemblies of these bolaamphiphiles and their binary self-assemblies with salmon sperm DNA were studied by AFM; temperature-dependent UV absorption, fluorescence, and circular dichroism spectroscopy; and gel electrophoresis. The binary self-assembly of dI(20) and salmon sperm DNA (dI(20)-DNA) had a nanosheet structure with a homogeneous thickness of about 6 nm and widths of several micrometers. Interestingly, an aqueous solution of the nanosheets showed a broad absorption band originating from the charge-transfer (CT) states of the nucleobase in the long-wavelength region (>300 nm), and the molar absorptivity per nucleobase was calculated to be approximately 150 times that of single-stranded (dT20 and dA20) and double-stranded (dT20-dA20) oligonucleotides. In addition, a continuous and broad emission band originating from CT excited states of the nucleobases was observed in the visible region. These observations indicate that CT states of the nucleobases were formed and stabilized in the supramolecular nanosheet and that bluish white light was emitted from CT excited states of the nucleobases.     

Probing the structure and dynamics of end-grafted flexible polymer chain layers by combined atomic force-electrochemical microscopy. Cyclic voltammetry within nanometer-thick macromolecular poly(ethylene glycol) layers

The combined atomic force-electrochemical microscopy (AFM-SECM) technique was used in aqueous solution to determine both the static and dynamical properties of nanometer-thick monolayers of poly(ethylene glycol) (PEG) chains end-grafted to a gold substrate surface. Approach of a microelectrode tip from a redox end-labeled PEG layer triggered a tip-to-substrate cycling motion of the chains' free ends as a result of the redox heads' oxidation at the tip and re-reduction at the substrate surface. As few as approximately 200 chains at a time could be addressed in such a way. Quantitative analysis of the data, in the light of a simple model of elastic bounded diffusion SECM positive feedback, gave access to the end-tethered polymer layer thickness and the end-to-end diffusion coefficient of the chains. The thickness of the grafted PEG layer was shown to increase with the chain surface coverage, while the end-to-end diffusion coefficient was found to be constant and close to the one predicted by Rouse dynamics. At close tip-substrate separation, slowing of the chains' motion, as a consequence of their vertical confinement within the tip-substrate gap, was observed and quantitatively modeled.     

Dynamics of electron transport by elastic bending of short DNA duplexes. Experimental study and quantitative modeling of the cyclic voltammetric behavior of 3'-ferrocenyl DNA end-grafted on gold

The dynamics of electron transport within a molecular monolayer of 3'-ferrocenylated-(dT)(20) strands, 5'-thiol end-grafted onto gold electrode surfaces via a short C2-alkyl linker, is analyzed using cyclic voltammetry as the excitation/measurement technique. It is shown that the single-stranded DNA layer behaves as a diffusionless system, due to the high flexibility of the ss-DNA chain. Upon hybridization by the fully complementary (dA)(20) target, the DNA-modified gold electrode displays a highly unusual voltammetric behavior, the faradaic signal even ultimately switching off at a high enough potential scan rate. This remarkable extinction phenomenon is qualitatively and quantitatively justified by the model of elastic bending diffusion developed in the present work which describes the motion of the DNA-borne ferrocene moiety as resulting from the elastic bending of the duplex DNA toward and away from the electrode surface. Its use allows us to demonstrate that the dynamics of electron transport within the hybridized DNA layer is solely controlled by the intrinsic bending elasticity of ds-DNA. Fast scan rate cyclic voltammetry of end-grafted, redox-labeled DNA layers is shown to be an extremely efficient method to probe the bending dynamics of short-DNA fragments in the submillisecond time range. The persistence length of the end-anchored ds-DNA, a parameter quantifying the flexibility of the nanometer-long duplex, can then be straightforwardly and accurately determined from the voltammetry data.     

Intercalating nucleic acids (INAs) containing insertions of 6H-indolo[2,3-b]quinoxaline

6H-Indolo[2,3-b]quinoxaline was studied as a covalently bound heteroaromatic intercalator. Six monomers were synthesized and incorporated into DNA oligonucleotides. Through a study of linker length dependence it was concluded that the linker between the oligo and the intercalator must consist of at least five C atoms in order to stabilize a DNA duplex. An intercalator with a 2′-deoxy-d-riboside linker to the oligo could also stabilize a DNA/RNA duplex, while (S)-4-(6-methylindolo[2,3-b]quinoxalin-3-ylmethoxy)-butane-1,2-diol was able to stabilize both DNA/DNA, DNA/RNA and a DNA/LNA duplex. Mismatch studies revealed a huge sensitivity to the C-C mismatch at the 5′-site of the intercalator.     

Accessing the dynamics of end-grafted flexible polymer chains by atomic force-electrochemical microscopy. Theoretical modeling of the approach curves by the elastic bounded diffusion model and Monte Carlo simulations. Evidence for compression-induced lateral chain escape

The dynamics of a molecular layer of linear poly(ethylene glycol) (PEG) chains of molecular weight 3400, bearing at one end a ferrocene (Fc) label and thiol end-grafted at a low surface coverage onto a gold substrate, is probed using combined atomic force-electrochemical microscopy (AFM-SECM), at the scale of approximately 100 molecules. Force and current approach curves are simultaneously recorded as a force-sensing microelectrode (tip) is inserted within the approximately 10 nm thick, redox labeled, PEG chain layer. Whereas the force approach curve gives access to the structure of the compressed PEG layer, the tip-current, resulting from tip-to-substrate redox cycling of the Fc head of the chain, is controlled by chain dynamics. The elastic bounded diffusion model, which considers the motion of the Fc head as diffusion in a conformational field, complemented by Monte Carlo (MC) simulations, from which the chain conformation can be derived for any degree of confinement, allows the theoretical tip-current approach curve to be calculated. The experimental current approach curve can then be very satisfyingly reproduced by theory, down to a tip-substrate separation of approximately 2 nm, using only one adjustable parameter characterizing the chain dynamics: the effective diffusion coefficient of the chain head. At closer tip-substrate separations, an unpredicted peak is observed in the experimental current approach curve, which is shown to find its origin in a compression-induced escape of the chain from within the narrowing tip-substrate gap. MC simulations provide quantitative support for lateral chain elongation as the escape mechanism.     

四甲基吡啶卟啉与核酸相互作用的稳态和时间分辨光谱

采用稳态吸收和荧光光谱、圆二色谱和皮秒时间分辨荧光光谱手段, 研究了5,10,15,20-四[4-(N-甲基吡啶)]卟啉(TMPyP4)与腺嘌呤(A)、鸟嘌呤(G)、胸腺嘧啶(T)和胞嘧啶(C)等4种碱基, 以及相应的核苷、核苷酸和单链DNA的结合能力和光谱学性质. 研究结果发现, 嘌呤与TMPyP4的结合能力比嘧啶的强. 对于某一碱基系列, 结合能力强弱顺序依次为: 碱基~核苷<核苷酸<单链DNA. 时间分辨荧光谱研究发现, 除鸟嘌呤外, 核酸和TMPyP4复合物的荧光动力学均含有快(1~2 ns)和慢(约10 ns)两个衰减过程, 它们分别是由激基复合体和环境极性对激发态TMPyP4分子的影响所致. 单链DNA能诱导TMPyP4产生诱导圆二色信号, 而单分子(碱基、核苷、核苷酸)则无此功能.     

2'-deoxycytidines carrying amino and thiol functionality: synthesis and incorporation by Vent (exo-) polymerase

[reaction: see text] The synthesis of 2'-deoxycytidine nucleosides bearing amino and thiol groups appended to the 5-position of the nucleobase via a butynyl linker is described. The corresponding triphosphates were then synthesized from the nucleoside and incorporated into oligonucleotides by Vent (exo(-)) DNA polymerase. The ability of Vent (exo(-)) polymerase to amplify oligonucleotides containing these functionalized cytidine derivatives in a polymerase chain reaction (PCR) was demonstrated for the amino-functionalized derivative.     

Oxepane nucleic acids: synthesis, characterization, and properties of oligonucleotides bearing a seven-membered carbohydrate ring

The synthesis and properties of oxepane nucleic acids (ONAs) are described. ONAs are sugar-phosphate oligomers in which the pentofuranose ring of DNA and RNA is replaced with a seven-membered (oxepane) sugar ring. The oxepane nucleoside monomers were prepared from the ring expansion reaction of a cyclopropanated glycal, 1, and their conversion into phosphoramidite derivatives allowed efficient assembly of ONAs on a solid support. ONAs (oT15 and oA15) were found to be much more resistant toward nuclease degradation than natural DNA (dT15 and dA15) in fetal bovine serum (FBS) after 24 h of incubation at 37 degrees C. ONAs also display several attributes in common with the naturally occurring DNA. For example, oT15 exhibited cross-pairing with complementary RNA to give a duplex (oT15/rA15) whose conformation evaluated by CD spectroscopy very closely matched that of the natural DNA/RNA hybrid (dT15/rA15). Furthermore, oT15 was found to elicit Escherichia coli RNase H-mediated degradation of the rA15 strand. When we compared the rates of RNase H-mediated degradation induced by 5- (furanose, dT15), 6- (2'-enopyranose, pT18), and 7-membered (oxepane, oT15) ring oligonucleotides at a temperature that ensures maximum duplex population (10 degrees C), the following trend was observed: dT15 > oT15 > pT18. The wider implications of these results are discussed in the context of our current understanding of the catalytic mechanism of the enzyme. The homopolymer oT15 also paired with its oxepane complement, oA15, to form a duplex structure that was different [as assessed by circular dichroic (CD) spectroscopy] and of lower thermal stability relative to the native dT15/dA15 hybrid. Hence, ONAs are useful tools for biological studies and provide new insights into the structure and function of natural and alternative genetic systems.     

Direct selective detection of genomic DNA from coliform using a fiber optic biosensor

Fiber optic biosensors operated in a total internal reflection format were prepared based on covalent immobilization of 25mer lacZ single-stranded nucleic acid probe. Genomic DNA from Escherichia coli was extracted and then sheared by sonication to prepare fragments of approximately 300mer length. Other targets included a 25mer fully complementary lacZ sequence, 100mer polymerase chain reaction (PCR) products containing the lacZ sequence at various locations, and non-complementary DNA including genomic samples from salmon sperm. Non-selective adsorption of non-complementary oligonucleotides (ncDNA) was found to occur at a significantly faster rate than hybridization of complementary oligomers (cDNA) in all cases. The presence of ncDNA oligonucleotides did not inhibit selective interactions between immobilized DNA and cDNA in solution. The presence of high concentrations of non-complementary genomic DNA had little effect on extent or speed of hybridization of complementary oligonucleotides. Detection of genomic fragments containing the lacZ sequence was possible in as little as 20 s by observation of the steady-state fluorescence intensity increase or by time-dependent rate of fluorescence intensity changes.     

Neomycin-neomycin dimer: an all-carbohydrate scaffold with high affinity for AT-rich DNA duplexes

A dimeric neomycin-neomycin conjugate 3 with a flexible linker, 2,2'-(ethylenedioxy)bis(ethylamine), has been synthesized and characterized. Dimer 3 can selectively bind to AT-rich DNA duplexes with high affinity. Biophysical studies have been performed between 3 and different nucleic acids with varying base composition and conformation by using ITC (isothermal calorimetry), CD (circular dichroism), FID (fluorescent intercalator displacement), and UV (ultraviolet) thermal denaturation experiments. A few conclusions can be drawn from this study: (1) FID assay with 3 and polynucleotides demonstrates the preference of 3 toward AT-rich sequences over GC-rich sequences. (2) FID assay and UV thermal denaturation experiments show that 3 has a higher affinity for the poly(dA)·poly(dT) DNA duplex than for the poly(dA)·2poly(dT) DNA triplex. Contrary to neomycin, 3 destabilizes poly(dA)·2poly(dT) triplex but stabilizes poly(dA)·poly(dT) duplex, suggesting the major groove as the binding site. (3) UV thermal denaturation studies and ITC experiments show that 3 stabilizes continuous AT-tract DNA better than DNA duplexes with alternating AT bases. (4) CD and FID titration studies show a DNA binding site size of 10-12 base pairs/drug, depending upon the structure/sequence of the duplex for AT-rich DNA duplexes. (5) FID and ITC titration between 3 and an intramolecular DNA duplex [d(5'-A(12)-x-T(12)-3'), x = hexaethylene glycol linker] results in a binding stoichiometry of 1:1 with a binding constant ～10(8) M(-1) at 100 mM KCl. (6) FID assay using 3 and 512 hairpin DNA sequences that vary in their AT base content and placement also show a higher binding selectivity of 3 toward continuous AT-rich than toward DNA duplexes with alternate AT base pairs. (7) Salt-dependent studies indicate the formation of three ion pairs during binding of the DNA duplex d[5'-A(12)-x-T(12)-3'] and 3. (8) ITC-derived binding constants between 3 and DNA duplexes have the following order: AT continuous, d[5'-G(3)A(5)T(5)C(3)-3'] > AT alternate, d[5'-G(3)(AT)(5)C(3)-3'] > GC-rich d[5'-A(3)G(5)C(5)T(3)-3']. (9) 3 binds to the AT-tract-containing DNA duplex (B* DNA, d[5'-G(3)A(5)T(5)C(3)-3']) with 1 order of magnitude higher affinity than to a DNA duplex with alternating AT base pairs (B DNA, d[5'-G(3)(AT)(5)C(3)-3']) and with almost 3 orders of magnitude higher affinity than a GC-rich DNA (A-form, d[5'-A(3)G(5)C(5)T(3)-3']).     

Recognition and detection of oligonucleotides in the presence of chromosomal DNA based on entrapment within conducting-polymer networks

Overlapping voltammetric signals, accrued from redox processes of nucleobases, do not permit discrimination between short oligonucleotides and chromosomal DNA molecules when conventional electrochemical techniques are used. This article describes a new genoelectronic route for discriminating between short oligonucleotides and chromosomal DNA, based on the polypyrrole (PPy) doping process. Such a route relies on the profound effect of short nucleic acid dopants upon the redox activity of PPy, and hence on the square-wave voltammetric signal of the polymer-modified electrode in a blank electrolyte solution. The electropolymeric growth of PPy thus serves for preferential accumulation (by doping) of short oligonucleotides. High selectivity is demonstrated for voltammetric measurements of oligo(dG)₂₀ and oligo(dT)₂₀ in the presence of otherwise interfering ss- and ds-DNA. The signals for the oligonucleotides are also not affected by a large excess of chloride or phosphate ions. The response of the new preconcentration (doping)–medium-exchange–voltammetric protocol is proportional to the concentration of the oligonucleotide dopant. Such a new recognition process, based on the doping of conducting-polymer networks, enhances the scope of electroanalysis of nucleic acids.     

Direct observation of guanine radical cation deprotonation in duplex DNA using pulse radiolysis

The dynamics of one-electron oxidation of guanine (G) base mononucleotide and that in DNA have been investigated by pulse radiolysis. The radical cation (G+*) of deoxyguanosine (dG), produced by oxidation with SO(4)-*, rapidly deprotonates to form the neutral G radical (G(-H)*) with a rate constant of 1.8 x 10(7) s(-1) at pH 7.0, as judged from transient spectroscopy. With experiments using different double-stranded oligonucleotides containing G, GG, and GGG sequences, the absorbance increases at 625 nm, characteristic of formation of the G(-H)*, were found to consist of two phases. The rate constants of the faster ( approximately 1.3 x 10(7) s(-1)) and slower phases ( approximately 3.0 x 10(6) s(-1)) were similar for the different oligonucleotides. On the other hand, in the oligonucleotide containing G located at the 5'- and 3'-terminal positions, only the faster phase was seen. These results suggest that the lifetime of the radical cation of the G:C base pair (GC+*), depending on its location in the DNA chain, is longer than that of free dG. In addition, the absorption spectral intermediates showed that hole transport to a specific G site within a 12-13mer double-stranded oligonucleotide is complete within 50 ns; that is, the rate of hole transport over 20 A is >10(7) s(-1).     

Molecular beacon probes of photodamage in thymine and uracil oligonucleotides

Molecular beacons (MB) are becoming more common as sequence-selective detectors of nucleic acids. Although they can easily detect single-base mismatches, they have never been used to directly detect DNA or RNA damage. To measure the degree of ultraviolet (UV) light damage in oligonucleotides, we report a novel MB approach for general detection of photoproducts in UV-irradiated rU17 and dT17 oligonucleotides. With monochromatic UV light irradiation at ca 280 nm under anoxic conditions, the oligonucleotide absorption decays with a single-exponential time constant of 123+/-1 min for rU17 and with double-exponential time constants of 78+/-0.5 min (99%) and 180+/-5 min (0.05%) for dT17 oligonucleotides. Under the same conditions, the MB fluorescence decays more quickly, with single-exponential time constants of 19+/-2 and 26+/-3 min for rU17 and dT17, respectively. Similar kinetics were observed with broadband UV light irradiation of oligonucleotides. The differences in the UV damage kinetics of dT17 and rU17 and their detection by absorption and fluorescence techniques will be discussed in the context of differential instabilities introduced in the nucleic acid-MB duplex by the different photoproducts formed.     

Sequence-specific control of azobenzene assemblies by molecular recognition of DNA

We investigated the molecular recognition between the amphiphile AzoAde, which is composed of azobenzene in the hydrophobic and adenine in the hydrophilic portion of the molecule, and oligonucleotides having a homogeneous base (dA30, dT30, dG30, and dC30) at the air-water interface. On the basis of the complementary base-pairing of DNA in the duplex, orderly arrangement of AzoAde on templated dT30 was examined using pi-A isotherm, UV-vis RAS, FT-IR RAS, and XPS measurements. Although there was little interaction between AzoAde and mismatched oligonucleotides (dA30, dG30, and dC30), AzoAde prepared on a dT30 subphase stoichiometrically assembled and interacted with dT30, subsequently forming a J-form assembly at the air-water interface. AFM observation of the LB films revealed the nanostructure of the J-formed AzoAde monolayer on the dT30 subphase as well as the domain structures of the H-formed monolayers on the other oligonucleotide subphases. Therefore, dT30 has a potential application as a template for assembling AzoAde at the air-water interface.