Factors influencing the electrokinetic injection of oligonucleotides in capillary gel electrophoresis when using laser‐induced fluorescence detection

Capillary gel electrophoresis (CGE) is a powerful tool for the analysis of oligonucleotides owing to its extraordinary resolving power. However, the only feasible injection mode for CGE, electrokinetic injection, can cause bias of the injected amount and thus reproducibility issues for CGE methods. Although the source of the bias in electrokinetic injection for analysis of small molecules by capillary zone electrophoresis has long been identified, there are very few studies on electrokinetic injection issues for biological molecules analyzed by CGE. In this study, we report three issues related to electrokinetic injection for oligonucleotides. First, the relationship between the injection amount and the sample solution resistance is not always linear for oligonucleotides, as has been observed for small molecules. Second, the injecting water prior to an oligonucleotide sample dramatically improves the reproducibility of both the injected amount and resolution through a ‘stacking‐like’ mechanism. Third, optimizing the gel concentration dramatically increases the amount of oligonucleotide that is injected into the column. Copyright © 2013 John Wiley & Sons, Ltd.     

Shotgun sequencing small oligonucleotides by nozzle-skimmer dissociation and electrospray ionization mass spectrometry

Nozzle-skimmer dissociation in combination with de novo sequencing was investigated as an approach for increasing the throughput of oligonucleotide analysis attainable by electrospray ionization mass spectrometry. An experimental method allowing for the sequential generation of precursor and fragment ion data during direct infusion of sample was developed. These data can then be used with readily available de novo sequencing software to characterize small oligonucleotides. When this approach was applied to mixtures of oligonucleotides, it was found that de novo sequencing becomes limited due to spectral congestion and overlapping oligonucleotide m/z dissociation product values. Self-packed C(18) microspray emitters were investigated as a means of reducing spectral complexity. It was found that such emitters allow for the analysis of oligonucleotide mixtures with minimal component overlap, and these emitters provide additional benefits of pre- concentrating and desalting the sample. These developments can provide a route for the more rapid characterization of ribonucleic acid endonuclease digestion mixtures.     

A comparative study on methods of optimal sample preparation for the analysis of oligonucleotides by matrix-assisted laser desorption/ionization mass spectrometry

Metal adducts (e.g., Na+ and K+) significantly hinder the analysis of oligonucleotides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Although a number of sample purification methods exist, to date no comparative study exists to determine the most efficient method for purifying oligonucleotides. The objective of this work was to perform such a study. Several different oligonucleotide samples were synthesized. Aliquots of these samples were then purposely contaminated with sodium acetate to generate representative contaminated (salted) oligonucleotide samples. A number of popular oligonucleotide purification techniques were then tested as to their effectiveness at removing Na+ from the salted samples. The effectiveness of Na+ removal was qualitatively assessed by comparing the MALDI mass spectra of the original sample, the salted sample, and the salted sample after purification. Micropipet tips packed with C18 reversed-phase packing material (e.g., Zip Tips) appear to be the most effective means of purifying the oligonucleotides investigated. Minidialysis was found to be an effective alternative for purifying higher molecular weight oligonucleotides (> 10,000 u).     

Using sol-gel/crown ether hybrid materials as desalting substrates for matrix-assisted laser desorption/ionization analysis of oligonucleotides

This study demonstrates the feasibility of using sol-gel/crown ether hybrid materials as sample substrates that reduce the intensity of the signals of sodium ion adducts of oligonucleotides during matrix-assisted laser desorption/ionization (MALDI) analysis. 2-Hydroxymethyl[15]crown-5 and 2-hydroxymethyl[18]crown-6 were added as dopants during the sol-gel process to generate desalting substrates for MALDI sample deposition. The results demonstrate that the sol-gel/crown ether hybrid materials effectively suppress the formation of sodiated oligonucleotides during MALDI analysis. The largest detectable molecular size for an oligonucleotide was a 100-mer, and the detection limit for an oligonucleotide 36-mer was ca. 20 fmol.     

GenoMass software: a tool based on electrospray ionization tandem mass spectrometry for characterization and sequencing of oligonucleotide adducts

The analysis of DNA adducts is of importance in understanding DNA damage, and in the last few years mass spectrometry (MS) has emerged as the most comprehensive and versatile tool for routine characterization of modified oligonucleotides. The structural analysis of modified oligonucleotides, although routinely analyzed using mass spectrometry, is followed by a large amount of data, and a significant challenge is to locate the exact position of the adduct by computational spectral interpretation, which still is a bottleneck. In this report, we present an additional feature of the in‐house developed GenoMass software, which determines the exact location of an adduct in modified oligonucleotides by connecting tandem mass spectrometry (MS/MS) to a combinatorial isomer library generated in silico for nucleic acids. The performance of this MS/MS approach using GenoMass software was evaluated by MS/MS data interpretation for an unadducted and its corresponding N‐acetylaminofluorene (AAF) adducted 17‐mer (5'OH‐CCT ACC CCT TCC TTG TA‐3′OH) oligonucleotide. Further computational screening of this AAF adducted 17‐mer oligonucleotide (5′OH‐CCT ACC CCT TCC TTG TA‐3′OH) from a complex oligonucleotide mixture was performed using GenoMass. Finally, GenoMass was also used to identify the positional isomers of the AAF adducted 15‐mer oligonucleotide (5′OH‐ATGAACCGGAGGCCC‐3′OH). GenoMass is a simple, fast, data interpretation software that uses an in silico constructed library to relate the MS/MS sequencing approach to identify the exact location of adduct on oligonucleotides. Copyright © 2012 John Wiley & Sons, Ltd.     

Formation and characterization of iron-oligonucleotide complexes with matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry

Iron-containing oligonucleotide negative ions can be generated by matrix-assisted laser desorption/ionization from a stainless steel target disk (by either defocusing the laser beam or by mixing iron salts such as FeCl3 with the matrix compound during the sample preparation). High resolution mass measurements reveal the presence of both Fe2+ (as M + Fe - 3H)- and Fe3+ (as M + Fe - 4H)- in the metal-oligonucleotide ions. The presence of Fe3+ is unexpected, and must involve replacement of protons from the nucleic bases or ribose groups as well as the phosphate groups of the oligonucleotides. Inspection of a range of small oligonucleotides and mononucleotides reveals that the presence of both Fe2+ and Fe3+ in the iron-biomolecule complexes is dependent on the number of acidic hydrogens that can be replaced in the oligonucleotide or nucleotide. Collisional dissociation of several metal-tetranucleotide ions revealed that the presence of the iron ion alters the fragmentation observed. The iron atom was observed to be present in all of the fragment ions, and, whenever possible, seemed to enhance the abundance of fragment ions containing both iron and a guanine nucleic base. These results suggest that iron may serve as a useful probe for characterizing phosphorylated biomolecules.     

Optimizing processing parameters for signal enhancement of oligonucleotide and protein arrays on ARChip Epoxy

Signal enhancement of oligonucleotide and protein arrays on ARChip Epoxy was achieved by optimizing chip processing parameters. The parameters investigated were fabrication, blocking and guide dot concentration, probe concentration and modification, print buffer, humidity during arraying, slide agitation, spot volume and spotter compatibility. The optimum oligonucleotide concentration was 20 microM, while the optimum protein concentration was 0.05 mg/ml. Amino-modified oligonucleotides were best able to be bound to the resin's epoxy groups at pH 8, whereas thiol-modified oligonucleotides displayed an optimum coupling value of pH 7. So as to avoid background (BG) contamination of probes around bright guide dots, the concentration of fluorescent guide dots was set to 1 muM. The most suitable print buffers for oligonucleotide arrays using both piezo- and contact-printing systems proved to be 3 x SSC/1.5 M betaine and commercial ArrayLink. When 0.01% monochlortriazinyl-beta-cyclodextrin sodium salt (MCT) was added, the hybridization signal doubled in strength as compared to plain buffer. The optimum print buffer for proteins was 0.1 N phosphate buffer, pH 8/10% glycerine. The optimum humidity for arraying oligonucleotides was 60% and for proteins 40%. Initially agitating slides for 15 min was found just as effective as agitating slides over the total hybridization period (2.5 h), and this resulted in a three times stronger signal.     

Characterization of noncovalent complexes formed between minor groove binding molecules and duplex DNA by electrospray ionization-mass spectrometry

The noncovalent complex formed in solution between minor groove binding molecules and an oligonucleotide duplex was investigated by electrospray ionization-mass spectrometry (ESI-MS). The oligonucleotide duplex formed between two sequence-specific 14-base pair oligonucleotides was observed intact by ESI-MS and in relatively high abundance compared to the individual single-stranded components. Only sequence-specific A:B duplexes were observed, with no evidence of random nonspecific aggregation (i.e., A:A or B:B) occurring under the conditions utilized. Due to the different molecular weights of the two 14-base pair oligonucleotides, unambiguous determination of each oligonucleotide and the sequence-specific duplex was confirmed through their detection at unique mass-to-charge ratios. The noncovalent complexes formed between the self-complementary 5′-dCGCAAATTTGCG-3′ oligonucleotide and three minor groove binding molecules (distamycin A, pentamidine, and Hoechst 33258) were also observed. Variation of several electrospray ionization interface parameters as well as collision-induced dissociation methods were utilized to characterize the nature and stability of the noncovalent complexes. The noncovalent complexes upon collisional activation dissociated into single-stranded oligonucleotides and single-stranded oligonucleotides associated with a minor groove binding molecule. ESI-MS shows potential for the study of small molecule-oligonucleotide duplex interactions and determination of small molecule binding stoichiometry.     

Biotin-enhanced fragmentation for direct deoxyribonucleic acid sequencing using matrix-assisted laser desorption/ionization mass spectrometry

Fragmentation of synthetic oligonucleotides under the influence of biotin was investigated using 3-hydroxypicolinic acid (3-HPA) as a matrix-assisted laser desorption/ionization (MALDI) matrix. Addition of biotin into the sample enhanced fragmentation of the oligonucleotide between bases. However, when the biotin was tagged to the 5'-terminus of the oligonucleotide, enhancements were observed not only in desorption/ionization efficiency but also in the fragmentation of molecular ions. The protonation/deprotonation process occurs on the tagged biotin is a possible reason for the enhancement in desorption/ionization. Site-specific backbone cleavage fragmentation patterns were observed. The sequences of oligonucleotides can be obtained from their fragment ions. The direct sequencing of a 5'-biotin-tagged 25-mer is demonstrated.     

Determination of triphenylphosphine oxide in active pharmaceutical ingredients by hollow-fiber liquid-phase microextraction followed by reversed-phase liquid chromatography

A versatile procedure has been developed and validated for the determination of triphenylphosphine oxide (TPPO) at low levels in various active pharmaceutical ingredients (APIs). This procedure incorporates the use of the novel hollow-fiber liquid-phase microextraction (LPME) for the measurement of this potential process-related impurity in aqueous solutions of APIs. A small volume (40 microL) of 1-octanol contained within a hollow polypropylene fiber is used for the extraction of TPPO from low pH aqueous API solutions. More than a 100-fold increase in the TPPO concentration is obtained without additional evaporation of the extract. Experimental parameters of the extraction procedure were investigated to optimize extraction efficiency and minimize sample matrix interference. Using HPLC/UV as the end analysis technique, the procedure was validated for TPPO in the concentration range of 3-16 microg/L with an API present at 1500 mg/L. The versatility of the method was demonstrated by applying the procedure to the analysis of APIs with different molecular structures. This simple LPME procedure is inexpensive and offers appropriate sensitivity for the intended use while providing several advantages over other analysis methods for pharmaceutical samples.     

Development of a quality control method for the characterization of oligonucleotides by capillary zone electrophoresis-electrospray ionization-quadrupole time of flight-mass spectrometry

A capillary zone electrophoresis-negative electrospray ionization-quadrupole time of flight-mass spectrometric method was developed for the characterization of oligonucleotides after synthesis, using model compounds. The major difficulty is the adduction of metal cations to the polyanionic backbone of the oligonucleotide sample, resulting in complex spectra and decreased sensitivity. Several approaches were investigated to circumvent this problem. Separation was performed in an ammonium carbonate buffer. During separation, the interfering metal ions were exchanged for ammonium ions, which are less tightly bound to the oligonucleotide when ionized. The influence of the addition of piperidine and imidazole or trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) to the running buffer for further reduction of cation adduction was investigated. Addition of CDTA to the buffer system resulted in a deconvoluted spectrum with very little adducts. On-line sample stacking proved vital to preconcentrate the samples. The pH and the concentration of the ammonium carbonate buffer as well as the electrophoresis voltage were optimized to achieve the best signal response for the oligonucleotides and a maximum reduction of the cation adducts as well as a short analysis time. Finally, the sheath liquid composition was examined for further improvement of the signal. The developed method was used to analyze different oligonucleotides (5000-9200 Da) in light of its use as a final quality control method for oligonucleotides in terms of purity and sequence homogeneity of the synthesized products. In all cases, very little adducts were observed in the deconvoluted spectra, and the relative errors of the measured molecular masses ranged from 3 to 35 ppm.     

Validation and application of a method for the determination of nicotine and five major metabolites in smokers' urine by solid-phase extraction and liquid chromatography-tandem mass spectrometry

An SPE-LC-MS/MS method was developed, validated and applied to the determination of nicotine and five major metabolites in human urine: cotinine, trans-3'-hydroxycotinine, nicotine-N-glucuronide, cotinine-N-glucuronide and trans-3'-hydroxycotinine-O-glucuronide. A 500 microL urine sample was pH-adjusted with phosphate buffer (1.5 mL) containing nicotine-methyl-d3, cotinine-methyl-d3 and trans-3'-hydroxycotinine-methyl-d3 internal standards. For the unconjugated metabolites, an aliquot (800 microL) of the buffered solution was applied to a 30 mg Oasis HLB-SPE column, rinsed with 2% NH4OH/H2O (3.0 mL) and H2O (3.0 mL) and eluted with methanol (500 microL). The eluate was analyzed isocratically (100% methanol) by LC-MS/MS on a diol column (50 x 2.1 mm). For the total metabolites, a beta-glucuronidase/buffer preparation (100 microL) was added to the remaining buffered solution and incubated at 37 degrees C (20 h). An aliquot (800 microL) of the enzymatically treated buffered solution was extracted and analyzed in the same manner. The conjugated metabolites were determined indirectly by subtraction. The quantitation range of the method (ng/mL) was 14-10,320 for nicotine, 15-9800 for cotinine and 32-19,220 for trans-3'-hydroxycotinine. The validated method was used to observe diurnal variations from a smoker's spot urine samples, elimination half-lives from a smoker's 24 h urine samples and metabolite distribution profiles in the spot and 24 h urine samples.     

Matrix effect on the analysis of oligonucleotides by using a mass spectrometer with a sonic spray ionization source.

Matrix or impurities remaining in a DNA sample solution after various sample treatment procedures may influence a subsequent DNA analysis. In this work, several matrices were investigated concerning their effects on the analysis of oligonucleotide by using an ion-trap mass spectrometer equipped with a sonic spray ionization source. Inorganic salts of sodium chloride and magnesium chloride depressed the signal intensity by about 50% when the content of the salts was about 10 microM. dNTPs and Taq showed more severe depression on the oligonucleotide. However, Tris, or (hydroxymethyl)aminomethane, intensified the signal intensity, if its content was within an appropriate range. When the content of Tris was about 500 microM, the signal intensity was enhanced by factors of 3 and 5 for the 6-mer and the 20-mer oligonucleotides, respectively. With the existence of Tris, matrix effects from the inorganic salts, dNTPs and Taq were reduced.     

Oligonucleotides with 2'-O-carboxymethyl group: synthesis and 2'-conjugation via amide bond formation on solid phase

An efficient method for synthesis of oligonucleotide 2'-conjugates via amide bond formation on solid phase is described. Protected oligonucleotides containing a 2'-O-carboxymethyl group were obtained by use of a novel uridine 3'phosphoramidite, where the carboxylic acid moiety was introduced as its allyl ester. This protecting group is stable to the conditions used in solid-phase oligonucleotide assembly, but easily removed by Pd(0) and morpholine treatment. 2'-O-Carboxymethylated oligonucleotides were then efficiently conjugated on a solid support under normal peptide coupling conditions to various amines or to the N-termini of small peptides to give products of high purity in good yield. The method is well suited in principle for the preparation of peptide-oligonucleotide conjugates containing an amide linkage between the 2'-position of an oligonucleotide and the N-terminus of a peptide.     

Interaction between 14mer DNA oligonucleotide and cationic surfactants of various chain lengths

In the recent genomic era, a novel gene silencing approach has been introduced based on the use of small synthetic oligonucleotides, such as antisense RNAs, siRNAs, to inhibit the expression of a specific target gene. Successful implementation of this methodology calls for the development of efficient systems to deliver small oligonucleotides into the cells using various natural and synthetic cationic agents. While extensive studies have focused on the interaction of various natural and synthetic cationic surfactants with long DNA, less attention has been paid to surfactant interaction with small oligonucleotides. In this study, the interaction between 14mer double stranded DNA and alkyltrimethylammonium bromides of C16 (cetyl, CTAB), C14 (tetradecyl, TTAB), and C12 (dodecyl, DTAB) chain lengths was investigated at different charge ratios by gel electrophoresis, ethidium bromide exclusion, circular dichroism, and UV melting. Our gel studies at 1 microM oligonucleotide concentration showed that CTAB, TTAB, and DTAB neutralize the oligonucleotides at a charge ratio (Z+/-) of 1, 14, and 50, respectively. At lower charge ratios, CTAB and TTAB interact with oligonucleotides, and the complexes show electrophoretic mobility shifts in the gel, while such mobility shifts were completely absent in the case of DTAB. UV melting experiments revealed that interaction with all three surfactants increased the thermostability of the oligonucleotide. The extent of thermal stabilization was highest in the case of CTAB, moderate in the case of TTAB, and extremely low in the case of DTAB. Oligonucleotides within fully neutralized complexes denatured at further higher temperatures, and again, stabilization was the highest in the case of CTAB followed by TTAB and DTAB, hence revealing that the oligonucleotides interacted more strongly with CTAB than with the other two surfactants. Ethidium bromide exclusion studies also supported our UV melting studies, confirming that CTAB binds most strongly to the oligonucleotide. CD titrations of oligonucleotides with increasing amounts of surfactants revealed common spectral patterns consisting of the progressive loss of CD signals for native helical DNA conformations. Overall, our results demonstrate that interaction between oligonucleotides and cationic surfactants, although qualitatively similar to long double stranded DNA, shows subtle differences that need to be understood to improve small oligonucleotide delivery into the cells by using common delivery agents that have been used to deliver long pieces of DNA.     

Capillary gas chromatography/negative ion chemical ionization mass spectrometry for the quantification of bile acids including 1 beta-hydroxylated and unsaturated bile acids in serum and urine

12 bile acids, including 1 beta-hydroxylated and unsaturated bile acids, have been quantified by capillary gas chromatography/negative ion chemical ionization mass spectrometry, using the trimethylsilyl(TMS) ether derivatives of bile acid pentafluorobenzyl(PFB) esters. The analysis time is 12 min and the minimum measurable amount is 100 fg for each bile acid. Bile acids in 200 microL of serum and 50 microL of urine from healthy human adults were measured. These small sample sizes enhance the practicality of using this method as a screening test for bile acids in the serum and urine of human infants, where small sample size is a major problem.     

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.     

Oligonucleotide sequencing using guanine-specific methylation and electrospray ionization ion trap mass spectrometry

This paper describes a novel method to map guanine bases in short oligonucleotides using a simple chemical modification reaction and subsequent analysis by electrospray ionization ion trap mass spectrometry (ITMS). In situ guanine-specific methylation followed by gas-phase fragmentation permits the determination of the positions of all guanine residues. Collision-induced dissociation (CID) of the monomethylated oligonucleotide strand promotes rapid depurination and further collision (MS3) of the apurinic oligonucleotide leads to preferential cleavage of the backbone at the site of depurination. The mass of the resulting complementary product ions verifies the position of each guanine base in the sequence. The utility of this methodology is demonstrated for oligonucleotide sequences up to 10 bases in length. In addition, this technique successfully illustrates the use of selective fragmentation for sequencing oligonucleotides by ITMS.     

Fungal pathogenic nucleic acid detection achieved with a microfluidic microarray device

Detection of polymerase chain reaction (PCR) products obtained from cultured greenhouse fungal pathogens, Botrytis cinerea and Didymella bryoniae has been achieved using a previously developed microfluidic microarray assembly (MMA) device. The flexible probe construction and rapid DNA detection resulted from the use of centrifugal pumping in the steps of probe introduction and sample delivery, respectively. The line arrays of the oligonucleotide probes were “printed” on a CD-like glass chip using a polydimethylsiloxane (PDMS) polymer plate with radial microfluidic channels, and the sample hybridizations were conducted within the spiral channels on the second plate. The experimental conditions of probe immobilization and sample hybridization were optimized, and both complementary oligonucleotides and PCR products were tested. We were able to achieve adequate fluorescent signals with a sample load as small as 0.5 nM (1 μL) for oligonucleotide samples; for PCR products, we achieved detection at the level of 3 ng.     

Assessment of the Full Compatibility of Copper(I)-Catalyzed Alkyne-Azide Cycloaddition and Oxime Click Reactions for bis-Labelling of Oligonucleotides

The conjugation of oligonucleotides with reporters is of great interest for improving their intrinsic properties or endowing new ones. In this context, we report herein a new procedure for the bis-labelling of oligonucleotides through oxime ligation (Click-O) and copper(I)-catalyzed alkyne–azide cycloaddition (Click-H). 5′-Azido and 3′-aldehyde precursors were incorporated into oligonucleotides, and subsequent coupling reactions through Click-O and Click-H (or vice versa) were successfully achieved. In particular, we exhaustively investigated the full compatibility of each required step for both tethering strategies. The results demonstrate that click Huisgen and click oxime reactions are fully compatible. However, whilst both approaches can deliver the targeted doubly conjugated oligonucleotide, the route involving click oxime ligation prior to click Huisgen is significantly more successful. Thus the reactions investigated here can be considered to be key elements of the chemical toolbox for the synthesis of highly sophisticated bioconjugates.