Identification of aberrant 2'-5' RNA linkage isomers by pellicular anion exchange chromatography

During chemical RNA synthesis, many undesired products may be formed. In addition to the "n-x" sequences, depurination products, and incompletely deprotected oligonucleotides, linkage isomers may form during condensation and/or deprotection of the synthetic products. Under acidic conditions, bond migration may alter normal 3'-5' diesters to aberrant 2'-5' diesters. This results in isomers that are difficult to identify by MS and LC-MS techniques because the isomers have identical masses. HPLC methods for identification of these isomers have not advanced because the isomers are not expected to exhibit differences in hydrophobicity that allow resolution by reversed-phase columns. Neither are changes in ionic interactions anticipated for these isomers that would allow resolution by ion exchange methods. We observed that chromatography on pellicular anion exchange phases, but not on porous anion exchange phases, completely resolves oligonucleotides with very slight conformation differences (e.g., DNA vs. RNA of identical sequence). Because incorporation of 2'-5' linkages in RNA will alter solution conformation slightly, we considered that this pellicular ion exchanger might also allow resolution of identical RNA sequences harboring aberrant 2'-5' linkages from those lacking aberrant 2'-5' linkages. Using the nonporous DNAPac PA200 column, we demonstrated a chromatographic procedure for resolving synthetic RNA with aberrant linkages from their normally linked counterparts. Under certain conditions, aberrant isomers are not completely resolved from those containing only normal linkages. Therefore, we also developed an independent linkage-confirming method using a 5'-3' exonuclease. This enzyme produces incomplete digestion products during digestion of synthetic RNA containing aberrant 2'-5' linkages, and these are readily resolved by DNAPac PA200 chromatography.     

Unusual chromatographic behavior of oligonucleotide sequence isomers on two different anion exchange HPLC columns

The retention behavior of the unmodified phosphodiester oligonucleotide sequence isomers was investigated on two different anion exchange columns: Biospher GMB 1000Q (based on DEAE-modified glycidyl methacrylate) and PolyWAX LP (based on silica with a crosslinked coating of linear polyethyleneimine). There was a notable difference in retention of oligonucleotides of the same composition but differing in the position of a single base. The most pronounced difference was observed between the oligonucleotides with the variable base in the end and in the center of the sequence. The use of either acetonitrile or 2-propanol as a mobile phase organic modifier did not markedly affect the retention time patterns. Prediction of the retention times of oligonucleotides must take into account the base position as well as identity. This is the first report of such a "same composition different sequence" effect, described for the short peptides, for synthetic oligonucleotides.     

Biosensor detection of triplex formation by modified oligonucleotides

Due to the instability of DNA oligonucleotides in biological solutions, antisense or antigene therapies aimed at modulation of specific gene expression will most likely require the use of oligonucleotides with modified backbones. Here, we examine the use of a surface plasmon resonance biosensor (BIAcore) to compare triplex-directed binding of modified oligonucleotides targeted to a region of the murine c-myc promoter. We describe optimization of experimental conditions to minimize nonspecific interactions between the oligonucleotides and the sensor chip surface, and the limitations imposed by certain backbones and sequence types. The abilities of pyrimidine oligonucleotides with various modified backbones to form specific triple helices with an immobilized hairpin duplex were readily determined using the biosensor. Modification of the third-strand oligonucleotide with RNA or 2(')-O-methyl RNA was found to enhance triplex formation, whereas phosphorothioate or phosphotriester substitutions abrogated it. A comparison of these results to DNase I footprinting experiments using the same oligonucleotides showed complete agreement between the two sets of data.     

Inactivation of viruses using novel protein A wash buffers

Low pH viral inactivation is typically performed in the eluate pool following the protein A capture step during the manufacturing of monoclonal antibodies and Fc‐fusion proteins. However, exposure to low pH has the potential to alter protein quality. To avoid these difficulties, novel wash buffers capable of inactivating viruses while antibodies or Fc‐fusion proteins were bound to protein A or mixed mode resins were developed. By equilibrating the column in high salt buffer (2 M ammonium sulfate or 3 M sodium chloride) after loading, the hydrophobic interactions between antibodies and protein A ligands were increased enough to prevent elution at pH 3. The ammonium sulfate was also found to cause binding of an antibody to a mixed mode cation exchange and a mixed mode anion exchange resin at pH values that caused elution in conventional cation and anion exchange resins (pH 3.5 for Capto Adhere and pH 8.0 for Capto MMC), indicating that retention was due to enhanced hydrophobic interactions. The potential of the 2 M ammonium sulfate pH 3 buffer, a 1 M arginine buffer, and a buffer containing the detergent LDAO to inactivate XMuLV virus when used as protein A wash buffers with a 1 hour contact time were studied. The high salt and detergent containing wash buffers provided about five logs of removal, determined using PCR, and complete combined removal and inactivation (> 6 logs), determined by measuring infectivity. The novel protein A washes could provide more rapid, automated viral inactivation steps with lower pool conductivities. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:406–413, 2015     

反义寡核苷酸药物癌泰得的定性分析方法研究

 癌泰得 (ACTCACTCAGGCCTCAGACT)为端粒酶表达抑制活性反义寡核苷酸 .为了探讨其定性检测手段 ,通过阴离子交换高效液相色谱和毛细管凝胶电泳分析方法 ,确定了该硫代寡核苷酸以及与其有关的短序列和部分未被硫代类似物的保留时间 ,并分析了不同混合物样品 .结果表明 ,阴离子交换高效液相色谱对硫代寡核苷酸骨架上的差异非常敏感 ,可很好地分离长度相同的硫代和未完全硫代类似物 ,并且随未被硫代磷酸基数目增加 ,保留时间依次缩短 .阴离子交换高效液相色谱对硫代寡核苷酸的长度不敏感 ,不能分离相差一个碱基的硫代寡核苷酸 .毛细管凝胶电泳可很好地分离长度相差一个碱基的硫代寡核苷酸 ,不能分离同长硫代和部分硫代寡核苷酸 .高效液相色谱结合毛细管凝胶电泳可有效地确定癌泰得的纯度和修饰程度 .     

High-performance liquid chromatography with concanavalin A immobilized by metal interactions on the stationary phase

Concanavalin A (Con A) was immobilized via metal interactions on macroporous, microparticulate silica support having covalently bound iminodiacetic acid functions (IDA-silica) chelated with Cu(II) at the surface. The amount of copper and of Con A in the column could readily be controlled by the conditions used for chelating the metal by IDA-silica and for immobilization of the lectin. The retention behavior of columns packed with the stationary phase did not change under a wide range of elution conditions, indicating no loss of immobilized lectin. However, the Con A proper could readily be removed from the column at pH 3.0 or together with Cu(II) by perfusion with EDTA at neutral pH. Columns containing Con A immobilized by this technique exhibited dual retention behavior for proteins, glycoproteins, and carbohydrates according to the pertinent glycan-lectin or protein-metal interactions. The glycoproteins, peroxidase and alpha 1-acid glycoprotein, were retained by the Con A moiety and eluted with eluents containing competing sugars, whereas the proteins, beta-lactoglobulin, alpha-chymotrypsinogen A, and ribonuclease A and B were retained by the chelated copper and were eluted and separated with eluents containing sodium chloride or borate. Binding constants of glycosides on the immobilized Con A were evaluated chromatographically and found to be one-third to two-thirds those reported in the literature on the basis of experiments in free solution.     

Thermolytic release of covalently linked DNA oligonucleotides and their conjugates from controlled-pore glass at near neutral pH

The functionalization of long chain alkylamine controlled-pore glass (CPG) with a 3-hydroxypropyl-(2-cyanoethyl)thiophosphoryl linker and its conversion to the support 7 has led to the synthesis of DNA oligonucleotides and their 3'- or (3',5')-conjugates. Indeed, CPG support 7 has been successfully employed in the synthesis of both native and fully phosphorothioated DNA 20-mers. Unlike conventional succinylated CPG supports, this distinctively functionalized support allows oligonucleotide deprotection and removal of the deprotection side products to proceed without releasing the oligonucleotide into the aqueous milieu. When freed from deprotection side products, the DNA oligonucleotide is thermolytically released from the support within 2 h under nearly neutral conditions (pH 7.2, 90 degrees C). The quality of these oligonucleotides is comparable to that of identical oligonucleotides synthesized from succinylated CPG supports in terms of shorter than full length oligonucleotide contaminants and overall yields. The versatility of the thermolytic CPG support 7 is further demonstrated by the synthesis of a DNA oligonucleotide (20-mer) and its conjugation with an azido and alkynyl groups at both 5'-and 3'-termini, respectively. The functionality of the (3',5')-heteroconjugated oligonucleotide 18 is verified by its circularization to the DNA oligonucleotide 19 under "click" chemistry conditions.     

High performance liquid chromatography of nucleotides. Major methods and their development

The separation of mono- and oligonucleotides possibilities by means of high performance ion-exchange, reversed-phase, so-called "ion-pair" and adsorption chromatography are studied. The influence of the eluent composition (solvent, salt) and pH on the retention, selectivity and resolution in reversed-phase and ion-exchange chromatography is investigated. The model of the hydrophobic-pair ion-exchange mechanism of ion-pair chromatography is considered. The conditions for analysis and preparative isolation of a desired component are optimized for selectivity, resolution and throughput. The methods for prediction of the optimal gradient elution program reasonable resolution at the desired retention time and for choosing the guard-column packing material are proposed. A design of the gradient for system and the version of slurry packing method for HPLC prolonged life-time columns are improved. The automatized analytical technique for determination of the oligonucleotide monomeric composition with two coupled microcolumns is described, that involves enzymatic digestion of an oligonucleotide followed by ion-exchange separation of the hydrolysate.     

Exposure to an Antisense Oligonucleotide Decreases Corticotropin-Releasing Factor Receptor Binding in Rat Pituitary Cultures

Abstract: Corticotropin-releasing factor (CRF) appears to integrate the endocrine, autonomic, immunologic, and behavioral responses of mammals to stress. To investigate further the role of CRF in the CNS, we have begun investigating the usefulness of "antisense knockdown" strategies directed against the CRF receptor using rat anterior pituitary gland primary cell cultures. The 15-mer antisense (5' CTG-CGG-GCG-CCG-TCC 3') and "scrambled" control (5' CGT-CCG-CGC-GCT-GCG 3') oligonucleotides were synthesized based on the rat CRF receptor sequence just downstream of the initiation codon. In each of four separate experiments, exposure to 10 µmol/L of antisense oligonucleotide for 40–67 h resulted in significant (17–36%) decreases in ¹²⁵I-ovine CRF binding to pituitary cells as compared with either control (no oligonucleotide) or 10 µmol/L of "scrambled" oligonucleotide. Moreover, compared with scrambled oligonucleotide, exposure to 10 µmol/L of antisense oligonucleotide, which produced a 22% decrease in CRF receptor binding, also resulted in a significant attenuation of the adrenocorticotrophic hormone response following a 30-min challenge with 100 pmol/L of CRF. Thus, CRF receptor antisense oligonucleotides apparently reduce functional expression of CRF receptors. This technique may be useful in studying the kinetics of CRF receptor production and the physiological functions of CRF receptors within the CNS.     

Determination of hydrophobicity of non-homologous series of anticancer drugs by reversed-phase high-performance liquid chromatography

The hydrophobiciy and specific hydrophobic surface area of 21 commercial anticancer drugs were determined by reversed-phase high-performance liquid chromatography on an octadecyl-silica column using methanol-water mixtures as eluents. Linear correlations were calculated between the log k′ values and the methanol concentration of the eluent, the intercept and slope were considered as the best estimation of the hydrophobicity and specific hydrophobic surface area. The relationship between retention characteristics and physicochemical parameters of drugs was evaluated by multivariate mathematical statistical methods, such as principal component analysis followed by two-dimensional non-linear mapping, varimax rotation and by cluster analysis. Anticancer drugs can be well separated by reversed-phase HPLC. Various multivariate mathematical statistical calculations indicate that the retention of the investigated drugs is mainly governed by hydrophobic and steric parameters. The results suggest that the use of principal component analysis followed by two-dimensional non-linear mapping is superior to cluster analysis for the evaluation of large retention data matrices.     

Rapid routes of synthesis of chemically reactive and highly radioactively labeled alpha- and beta-oligonucleotide derivatives for in vivo studies

Development of the antisense oligonucleotide strategy for the regulation of gene expression in vivo poses several problems: the stability of oligonucleotides toward intracellular nucleases, labeling of oligonucleotides with high specific radioactivity, improvements of penetration of oligonucleotides into living cells, and enhancement of antisense action by coupling of chemically active groups. In the present paper synthesis of highly radioactively labeled [32P]- and [35S]oligonucleotide derivatives is described starting from both natural (beta) and nuclease-resistant (alpha) anomers of oligonucleotides. Conditions for preparative phosphorylation and thiophosphorylation suitable for oligonucleotides of various lengths, base composition, and anomeric forms were established. The stability of the phosphoramide bond under in vivo experimental conditions was checked. The methods of terminal phosphate chemical activation and terminal thiophosphate alkylation were applied to synthesize oligonucleotides equipped with hydrophobic, intercalating, alkylating, and photoactivatable groups. In the case of porphyrin-oligonucleotide conjugates, a series of new monofunctional porphyrin derivatives bearing a free aliphatic amino group was developed.     

Molecular recognition of oligonucleotides and plasmid DNA by l‐methionine

The present study explores the effect of oligonucleotide composition on the mechanism of retention to l ‐methionine agarose support by chromatography and saturation transfer difference (STD)‐nuclear magnetic resonance (NMR) techniques. All chromatographic experiments were performed using 1.5 M (NH₄)₂SO₄. The binding profiles obtained by chromatography show that oligonucleotides with thymine had the highest retention time. In general, the larger homo‐oligonucleotides are more retained to the l ‐methionine agarose support. Moreover, the study with hetero‐oligonucleotides confirms that the presence of guanine reduces the retention on the l ‐methionine chromatographic support. These results are in accord with STD‐NMR experiments, which show that the strongest signals were observed for the methyl group of thymine, and no STD signals were observed for the guanosine protons. Finally, the retention behaviour of linear plasmid DNA (pDNA) with different sizes and base composition (2.7‐kbp pUC19, 6.05‐kbp pVAX1‐LacZ, 7.4‐kbp pVAX1‐LacZgag and 14‐kbp pcDNA‐based plasmid) was also evaluated by chromatography. The results indicate that the underlying mechanism of retention involves not only hydrophobic interactions but also other elementary interactions responsible for the biorecognition of pDNA molecules by l ‐methionine ligands. Copyright © 2014 John Wiley & Sons, Ltd.     

Application of CEC procedures for the analysis of synthetic peptides: characterization of linear immunogenic peptides that mimic a HIV-1 gp120 epitope.

In this study, we describe the application of a new analytical procedure based on capillary electrochromatographic(CEC) techniques for the characterization of different basic and acidic peptides using isocratic eluent conditions containing acetonitrile and ammonium acetate buffers of different molarities between pH 3.8 and 5.2. In particular,10 immunogenic peptide analogs with isoelectric points ranging from 3.7 to 10.1 were investigated; nine of these peptides, 1-9, were truncated analogs of the parent peptide, 10, which is a peptidomimetic related to a HIV-1 gp120 epitope. Several of these peptides have the propensity to form alpha-helical secondary structures in solution. Electrochromatographic separations of these peptides were achieved with packed fused silica capillaries(25 cm packed length, 100 microm i.d.) containing 3 microm n-octadecylsilica particles. The influence of temperature on the CEC elution behavior of these peptides, as well as the impact of changes in the eluent composition, e.g. pH, buffer concentration and acetonitrile content, were examined. The results confirm that improvements in the resolution and analysis of synthetic peptides by CEC procedures result from the increase inelectroosmotic flow (EOF) as the temperature is increased.These findings emphasize the dominant influence of the temperature-dependent viscosity parameter, eta, on the EOF and thus on peptide resolution in CEC. Moreover, these investigations have shown that eluent properties can be specifically chosen to favor either electrophoretic mobility or chromatographic retention, with the overall CEC selectivity peptides of different sequence or composition reflecting the summated contributions from both separation mechanisms. Over the pH range 4.0-5.0, and using eluents with ionic strengths ranging from 6.2 to 15 mM ammonium acetate but containing a fixed volume fraction, psi, of acetonitrile above psi = 0.40, the CEC retention behavior of peptides 1-10 correlated with a linear relationship linking the retention coefficient, kappta(cec), and the differential frictional size-to-mass ratio parameter, Xi(fric), of these peptides. However, using eluents with a low acetonitrile content and low pH values, linear correlations were also observed between the incremental retention coefficient, Delta(Kappa)cec, and the product term [-0.66(Delta(Sigma[Xn]) log(Mi/Mj)], which links the difference in intrinsic hydrophobicities and molecular masses of two peptides, Pi and Pj. This study thus demonstrates the power of CEC procedures in the analysis of synthetic bioactive peptides and provides a general experimental framework to evaluate,using CEC procedures, the influence of the key molecular attributes of peptides on their structure-retention dependencies.Finally, these studies provide additional, practical insights into the use of CEC procedures for the analysis, resolution and biophysical characterization of closely related peptide analogs derived from solid-state peptide synthesis under conditions of different eluent composition or temperature.     

Separation of synthetic oligonucleotide dithioates from monothiophosphate impurities by anion-exchange chromatography on a mono-q column

A method using a strong anion-exchange liquid-chromatography column, Mono-Q, has been developed for high-resolution analysis and purification of oligonucleotide dithioates, which were synthesized by an automated, solid-phase, phosphorothioamidite chemistry. High-resolution separation of oligonucleotide phosphorodithioates from monothiophosphate impurities was obtained. High-resolution separation was also demonstrated at pH 8. The separation of oligonucleotide dithioates was found to be linearly dependent on the number of sulfurs for the same sequence length. Thiocyanate, SCN-, as eluting anion, can be used to purify oligonucleotides containing a high percentage of phosphorodithioate linkages in lower salt concentrations and provides better separation than chloride as eluting anion.     

Fluorescent dye phosphoramidite labelling of oligonucleotides.

A series of fluorescein phosphoramidites (FAM) have been synthesized for use on automated DNA synthesizers. After coupling of the FAM reagents to the 5' hydroxyl of the oligonucleotide on the DNA synthesizer, the excess reagent is removed by washing the solid support. The dye, and its linkage to the oligonucleotide, are stable during the conditions of DNA synthesis and cleavage/deprotection conditions. Purification is attained with the OPC (Oligonucleotide Purification Cartridge), a polystyrene based affinity matrix, which selectively retains hydrophobic oligonucleotide conjugates. Analysis by MicroGel capillary electrophoresis effectively separates fluorescent dye labelled oligonucleotides from unlabelled products.     

Oligonucleotide analysis by anion exchange HPLC.

The ability to resolve and purify synthetic oligonucleotides by high performance anion exchange chromatography was evaluated using two wide pore polymeric HPLC matrices. The materials used are rigid macroporous copolymers which have a fully quaternised polyethyleneimine coating to provide a strong anion exchange, quaternary amine, functionality. Oligomers of poly(rA), poly(rC) and RNA produced by alkaline hydrolysis of the polymers were chromatographed to evaluate the selectivity of the system prior to the analysis of synthetic oligonucleotides produced using a commercial oligonucleotide synthesizer.     

Fluorescence resonance energy transfer between donor-acceptor pair on two oligonucleotides hybridized adjacently to DNA template

We use fluorescein as the energy donor and rhodamine as the acceptor to measure the efficiency of fluorescence resonance energy transfer (FRET) in a set of hybridized DNA constructs. The two fluorophores are covalently attached via linkers to two separate oligonucleotides with fluorescein at the 3' end of one oligonucleotide and rhodamine at the 5' end or in the middle of another nucleotide. For the FRET analysis both fluorophore-labeled oligonucleotides are hybridized to adjacent sections of the same DNA template to form a three-component duplex with a one base gap between the two labeled oligonucleotides. A similar configuration is implemented for a quantitative real-time polymerase chain reaction (PCR) with LightCycler technology, where a 1-5 base separation between donor and acceptor is recommended to optimize energy transfer efficiencies. Our constructs cover donor-acceptor separations from 2 to 17 base pairs (approximately 10-70 A). The results show that, when the two fluorophores are located at close distances (less than 8 base separation), FRET efficiencies are above 80%, although there may be ground-state interactions between fluorophores when the separation is under about 6 bases. Modeling calculations are used to predict the structure of these three-component constructs. The duplex mostly retains a normal double helical structure, although slight bending may occur near the unpaired base in the DNA template. Stable and reproducible energy transfer is also observed over the distance range investigated here in real-time thermal cycling. The study identifies important parameters that determine FRET response in applications such as real-time PCR.