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.     

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.     

Capillary gel electrophoresis of oligonucleotides using polymer solutions

Summary Capillary gel electrophoresis (CGE) has been recognized as an effective method for the analysis of oligonucleotides. CGE using polymer solutions is especially useful and effective compared with that using crosslinked gels, because of easy change of media. Replacement of media leads to the reproducible separation of analytes. We have investigated CGE analysis of oligonucleotides of less than 20 bases employing various kinds of polymers. Polyacrylamide, dextrin, dextran, pullakin, and poly(ethylene glycol) were used as sieving matrixes at concentrations of 0–30 %. Polydeoxythymidylic acids [p(dT)_11–20] were used as a test sample. These small oligonucleotides were successfully resolved on the basis of their base number by CGE using some of these polymer solutions. In particular, dextran was found to be effective and baseline separation was observed when a 30 % dextran solution was employed. Some validations such as linearity and reproducibility were also established and this method was found to be an adequate quality control method for small oligonucleotides. Finally, CGE using a 30 % dextran solution was successfully applied to impurity profiling of some synthetic oligonucleotides.     

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.     

Recognition and strand displacement of DNA oligonucleotides by peptide nucleic acids (PNAs). High-performance ion-exchange chromatographic analysis

Peptide nucleic acids (PNAs) are oligonucleotide mimics containing a pseudopeptide chain, which are able to bind complementary DNA tracts with high affinity and selectivity. Two mixed-sequence PNA undecamers (1 and 2) were synthesized and their double-stranded adducts with the complementary oligonucleotides (3 and 4) were revealed by the appearance of the corresponding peak in anion-exchange HPLC. A DEAE column was used and elution was performed with aqueous Tris buffer (pH 8) and an ionic strength gradient (0-0.5 M NaCl). The same effect was not observed with non-complementary oligonucleotides. The stability of the PNA-DNA adducts under the conditions used in the chromatographic system was studied as a function of temperature. Furthermore, in competition experiments double-stranded oligonucleotides were challenged by a PNA complementary to one strand: the formation of the PNA-DNA hybrid and the displacement of the non-complementary strand were observed with high specificity. The results suggest a possible use of ion-exchange HPLC for studying PNA-DNA interactions, and indicate the efficiency of PNA probes in the chromatographic analysis of DNA.     

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.     

Ion-pair reversed-phase high-performance liquid chromatography analysis of oligonucleotides: retention prediction

An ion-pair reversed-phase HPLC method was evaluated for the separation of synthetic oligonucleotides. Mass transfer in the stationary phase was found to be a major factor contributing to peak broadening on porous C18 stationary phases. A small sorbent particle size (2.5 microm), elevated temperature and a relatively slow flow-rate were utilized to enhance mass transfer. A short 50 mm column allows for an efficient separation up to 30mer oligonucleotides. The separation strategy consists of a shallow linear gradient of organic modifier, optimal initial gradient strength, and the use of an ion-pairing buffer. The triethylammonium acetate ion-pairing mobile phases have been traditionally used for oligonucleotide separations with good result. However, the oligonucleotide retention is affected by its nucleotide composition. We developed a mathematical model for the prediction of oligonucleotide retention from sequence and length. We used the model successfully to select the optimal initial gradient strength for fast HPLC purification of synthetic oligonucleotides. We also utilized ion-pairing mobile phases comprised of triethylamine (TEA) buffered by hexafluoroisopropanol (HFIP). The TEA-HFIP aqueous buffers are useful for a highly efficient and less sequence-dependent separation of heterooligonucleotides.     

Photoelectron spectroscopy of isolated multiply negatively charged oligonucleotides

Ultraviolet photoelectron spectroscopy in an ion beam was used to investigate the electronic properties of isolated DNA oligonucleotides [dA(5)-4H](4-) and [dT(5)-4H](4-), carrying four excess negative charges. We find the fourth adiabatic electron affinity to be slightly negative for [dA(5)-4H](4-), while it is positive for [dT(5)-4H](4-). This implies a significant influence of the base composition on energetics, which is in turn relevant for analytic applications and also for charge transport properties.     

Synthesis and biophysical characterization of oligonucleotides containing a 4'-selenonucleotide

The first synthesis of oligonucleotides containing 4'-selenium-modified ribonucleotides (4'-Se-rN) is described. Four sequences containing 4'-Se-rT were successfully synthesized and compared with DNA and RNA oligonucleotides containing a dT, rT, or LNA insert in place of the 4'-Se-rT. The 4'-Se-rT behaved more like rT than dT in its effects on binding affinity, despite the DNA-like structure previously observed for the nucleoside, suggesting that a conformational switch occurs upon incorporation into an oligonucleotide. Incorporation of 4'-Se-rT into A-RNA and hybrid duplexes led to increased binding affinity, while incorporation into B-DNA destabilized the duplex to the same extent as an rT nucleotide.     

A sub-two minutes method for monoclonal antibody-aggregate quantification using parallel interlaced size exclusion high performance liquid chromatography

In process development and during commercial production of monoclonal antibodies (mAb) the monitoring of aggregate levels is obligatory. The standard assay for mAb aggregate quantification is based on size exclusion chromatography (SEC) performed on a HPLC system. Advantages hereof are high precision and simplicity, however, standard SEC methodology is very time consuming. With an average throughput of usually two samples per hour, it neither fits to high throughput process development (HTPD), nor is it applicable for purification process monitoring. We present a comparison of three different SEC columns for mAb-aggregate quantification addressing throughput, resolution, and reproducibility. A short column (150 mm) with sub-two micron particles was shown to generate high resolution (~1.5) and precision (coefficient of variation (cv)<1) with an assay time below 6 min. This column type was then used to combine interlaced sample injections with parallelization of two columns aiming for an absolute minimal assay time. By doing so, both lag times before and after the peaks of interest were successfully eliminated resulting in an assay time below 2 min. It was demonstrated that determined aggregate levels and precision of the throughput optimized SEC assay were equal to those of a single injection based assay. Hence, the presented methodology of parallel interlaced SEC (PI-SEC) represents a valuable tool addressing HTPD and process monitoring.     

Extent of formation of a dimeric adenine photoproduct in polynucleotides and DNA

Quantum yields are reported for the formation of a dimeric adenine photoproduct, A = A, in adenine homopolymers and DNA irradiated at 254 nm. The A = A content of irradiated samples was assayed by using reversed-phase HPLC to isolate the 4,6-diamino-5-guanidinopyrimidine (DGPY) which is produced from A = A on acid hydrolysis. Acid hydrolysates derived from DNA radiolabelled with [14C] 2'-deoxyadenosine were spiked with unlabelled DGPY before fractionation on HPLC and the recovered material was further purified by chromatography on Sephadex G-10 followed by co-crystallization with DGPY sulphate. Although A = A is formed with a relatively high quantum yield of 1.6 X 10(-3) mol einstein-1 in single-stranded poly(dA) the photoaddition reaction is strongly quenched in base-paired poly(dA).poly(dT) and undetectable in poly(rA).poly(dT). Respective quantum yields of 6 X 10(-5) and 9 X 10(-6) were estimated for the formation of A = A in single- and double-stranded E. coli DNA implying that the photoproduct has very limited biological significance. From studies with d(ApG), d(GpA), ApG, GpA, d(A)20 and d(A4G)4 it is concluded that adjacent guanine and adenine bases do not form a photoadduct analogous to A = A and also that guanine residues have no local or long-range quenching effect on photodimerization within A-A doublets.     

pH‐Independent Recognition of the dG ⋅ dC Base Pair in Triplex DNA: 9‐Deazaguanine N7‐(2′‐Deoxyribonucleoside) and Halogenated Derivatives Replacing Protonated dC

Triplex-forming oligonucleotides (TFOs) containing 9-deazaguanine N⁷-(2′-deoxyribonucleoside) 1a and halogenated derivatives 1b,c were synthesized employing solid-phase oligonucleotide synthesis. For that purpose, the phosphoramidite building blocks 5a – c and 8a – c were synthesized. Multiple incorporations of 1a – c in place of dC were performed within TFOs, which involved the sequence of five consecutive 1a – c ⋅ dG ⋅ dC triplets as well as of three alternating 1a – c ⋅ dG ⋅ dC and dT ⋅ dA ⋅ dT triplets. These TFOs were designed to bind in a parallel orientation to the target duplex. Triplex forming properties of these oligonucleotides containing 1a – c in the presence of Na⁺ and Mg²⁺ were studied by UV/melting-curve analysis and confirmed by circular-dichroism (CD) spectroscopy. The oligonucleotides containing 1a in the place of dC formed stable triplexes at physiological pH in the case of sequence of five consecutive 1a ⋅ dG ⋅ dC triplets as well as three alternating 1a – c ⋅ dG ⋅ dC and dT ⋅ dA ⋅ dT triplets. The replacement of 1a by 9-halogenated derivatives 1b,c further enhanced the stability of DNA triplexes. Nucleosides 1a – c also stabilized duplex DNA.     

Behaviour of nucleotides and oligonucleotides in potentiometric HPLC detection

Potentiometric HPLC detection was studied of mononucleotides (UMP, AMP, UDP, ADP, CTP, UTP, GTP, ATP) and oligonucleotides (a synthetic mixture d(T)_12–18 5′OH, a mixed 21-mer, 33-mer, and 60-mer). Coated-wire electrodes were used. The coatings were of the liquid membrane type containing PVC, DOS and synthetic macrocyclic amine- and podand ureum receptors. Electrodes based on these receptors gave very sensitive responses to triphosphate nucleotides and to oligonucleotides. The molar response of the oligonucleotides was related to their molar mass. The HPLC system consisted of a reversed phase column eluted with a phosphate buffer, triethylammoniumacetate (TEAA), and an acetonitrile gradient. The sensitive potentiometric response of these highly charged ions is discussed.     

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.     

The use of multidimensional chromatography for the isolation of synthetic oligodeoxyribonucleotides on a preparative scale

Summary A critical step in the chemical preparation of oligonucleotides is the chromatographic purification of the deprotected oligomers. In case of large quantities of reaction products, the oligonucleotides are first enriched on a QAE-Sephadex column at low pressure. The obtained fractions are then purified by multidimensional chromatography making use of three independent physical properties of the solutes: molecular size, ionic net charge and hydrophobicity. In the first dimension size exclusion chromatography (Sephadex G-15) is used. In the second dimension the high molecular weight fraction from the size exclusion chromatography is applied to a HPLC ion-exchange column (Partisil-10 SAX). Usually the last peak is collected and transferred to a HPLC reversed phase column (Nucleosil C18) where the components are separated according to their hydrophobicity in the third dimension. The efficiency of this multi-dimensional chromatographic procedure is demonstrated by the unequivocal fingerprints after radioactive labelling of the isolated oligonucleotides. Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1984     

Non-contact production of oligonucleotide microarrays using the highly integrated TopSpot nanoliter dispenser

For the first time we report on the production of oligonucleotide microarrays using a highly parallel and highly integrated, pressure driven TopSpot nanoliter dispenser. The system enables non-contact printing of different media like oligonucleotides, DNA or protein solutions. We optimized the printing buffer needed for oligonucleotides microarrays production with respect to two major aspects: microfluidical optimum for droplet dispensing and biochemical coupling efficiency on different commercially available microarray slides. Coefficient of variations (CVs) of generated spot diameters were measured to be smaller than 1% within one single dispensing nozzle and smaller than 1.5% within all 24 parallel nozzles of the printhead for all printing buffers used. No carry-over and no cross-talk was found, in extensive experiments with oligonucleotides. Optimized printing buffer compositions and concentrations for oligonucleotide microarrays were found, as well as optimized coupling protocols. Furthermore, buffers and protocols were adapted to a host of different microarray slides used. With this system, prime critical points of microarray production are solved, leading to high quality high throughput microarray fabrication.     

Characterization of the adsorption of oligonucleotides on mercaptopropionic acid-coated CdSe/ZnS quantum dots using fluorescence resonance energy transfer

Semiconductor quantum dots (QDs) coated with thioalkyl acid ligands are often used as probes and reporters for nucleic acid sensing, or protein sensing using aptamers, and are also potential vectors for gene delivery. In such applications, the interactions that potentially lead to the adsorption of oligonucleotides onto the surface of colloidal QDs are an important consideration. To explore such interactions, fluorescence resonance energy transfer (FRET) between QDs and oligonucleotides labeled with a fluorescent dye was used to identify and characterize a set of conditions that favor strong adsorption on 3-mercaptopropionic acid (MPA)-coated CdSe/ZnS QDs. Adsorption curves and competitive binding experiments were used to determine that the order of affinity for nucleobase adsorption was dC>dA≥dG?dT. The length of the oligonucleotide sequence was also important, with an 80-mer sequence adsorbing more strongly than its 20-mer analog. Adsorption decreased with increasing pH and corresponded to the ionization of the carboxylic acid groups of the MPA ligands. Increased ionic strength partially offsets ligand ionization and increased the extent of adsorption. The interaction between QDs and oligonucleotides was labile, with increases in adsorption at lower concentrations of oligonucleotide and with an increasing number of oligonucleotides per QD. The results were consistent with a hydrogen-bonding model for adsorption, where neutral thioalkyl acid ligands interact favorably with nucleobases and ionized ligands resist adsorption.     

Demonstration of the capabilities of a parallel high performance liquid chromatography tandem mass spectrometry system for use in the analysis of drug discovery plasma samples.

There is a continuing need for increased throughput in the evaluation of new drug entities in terms of their pharmacokinetic (PK) parameters. This report describes an alternative procedure for increasing the throughput of plasma samples assayed in one overnight analysis: the use of parallel high performance liquid chromatography (HPLC) combined with tandem mass spectrometry (parallel LC/MS/MS). For this work, two HPLC systems were linked so that their combined effluent flowed into one tandem MS system. The parallel HPLC/APCI-MS/MS system consisted of two Waters 2690 Alliance systems (each one included an HPLC pump and an autosampler) and one Finnigan TSQ 7000 triple quadrupole mass spectrometer. Therefore, the simultaneous chromatographic separation of the plasma samples was carried out in parallel on two HPLC systems. The MS data system was able to deconvolute the data to calculate the results for the samples. Using this system, 20 compounds were tested in one overnight assay using the rapid rat PK screening model which includes a total of 10 standards plus samples and two solvent blanks per compound tested. This application provides an additional means of increasing throughput in the drug discovery PK assay arena; using this approach a two-fold increase in throughput can be achieved in the assay part of the drug discovery rat PK screening step.     

天冬酰胺合成酶B抑制剂高效液相色谱筛选方法的建立与应用

建立了一种快速、高效测定天冬酰胺合成酶B(AS-B)酶活性的反相高效液相色谱法(RP-HPLC)。酶反应体系中的氨基酸经2,4-二硝基氟苯(DNFB)柱前衍生,通过RP-HPLC测定酶反应体系前后底物及产物的变化来分析酶的活性。采用的色谱柱为Agilent C18柱(250 mm×4.6 mm,5 μm),以50 mmol/L醋酸钠缓冲液(pH 6.2)-乙腈(体积比为15:85)为流动相,流速为1.0 mL/min,柱温为30 ℃,检测波长365 nm,于6 min内实现了各组分的基线分离。通过该方法测定反应动力学参数进行AS-B的抑制定量分析。将已知AS-B抑制剂L-谷氨酸-γ-甲酯作用于酶反应体系,测得的抑制剂的抑制常数与文献值相接近,证明该方法可用于AS-B抑制剂的筛选。     

Packings and stationary phases for biopolymer separations by HPLC

Summary Packings and stationary phases applied to high resolution separations of proteins, enzymes, and nucleic acids must satisfy a series of distinct criteria that are different from those usually required by HPLC of low molecular weight non-biologically active analytes. These requirements have been met through substantial improvements in classical gel media together with novel developments in silica supports, and have led to a family of products with tailor-made and reproducible properties. Supports consisting of cross-linked organic gels, and inorganic materials (mostly silicas) are now available with graduated particle sizes, pore sizes, porosities and surface areas as well as non-porous beads. A whole range of stationary phases, such as reversed phase, hydrophobic interaction, ion exchanger and affinity packings, were designed for application as chemical sensors for biopolymer recognition in adsorptive chromatography. The phase systems are operated in the gradient mode, giving high resolution and high peak capacities. In addition, aqueous liquid-liquid partitioning systems have been developed for the fractionation of proteins and nucleic acids. Size exclusion media complete the set of HPLC variants enabling a discrimination of proteins according to their size and shape in an isocratic elution mode. Basically, protein purification and isolation is a multistage process where-by the HPLC variants are combined in a logistic sequence, utilizing the different selectivities of the phase systems and thus maximising resolution, speed and throughput.