Improved oligonucleotide sequencing by alkaline phosphatase and exonuclease digestions with mass spectrometry

The combination of exonuclease digestion and mass spectrometry has been widely used for sequencing oligonucleotides. During an exonuclease digestion, rapid buildup in the concentration of nucleotides produces strong signal of nucleotide cluster ions in electrospray ionization-mass spectrometry, especially for oligonucleotides with greater than 25 bases. This leads to poor signal/noise ratio in the reconstructed molecular weight spectra of late digestion products due to artifact peaks from nucleotide cluster ions. Here we report a procedure that eliminates the effect of the cluster ions. In this method, alkaline phosphatase is added with snake venom phosphodiesterase to the oligonucleotide solution to convert the interfering nucleotides into noninterfering nucleosides, and the collision-induced dissociation spectrum of the dimeric oligonucleotide at the end of the digestion is obtained to determine the sequence of the last two bases at the 5'-terminus of the oligonucleotide. With this approach, the signal/noise ratio of the reconstructed molecular weight spectrum is greatly improved for relatively large oligonucleotides, and only a single digestion is needed for sequencing.     

On the use of ESI‐QqTOF‐MS/MS for the comparative sequencing of nucleic acids

The usability of a quadrupole—quadrupole—time‐of‐flight (QqTOF) instrument for the tandem mass spectrometric sequencing of oligodeoxynuleotides was investigated. The sample set consisted of 21 synthetic oligodeoxynucleotides ranging in length from 5 to 42 nucleotides. The sequences were randomly selected. For the majority of tested oligonucleotides, two or three different charge states were selected as precursor ions. Each precursor ion was fragmented applying several different collision voltages. Overall 282 fragment ion mass spectra were acquired. Computer‐aided interpretation of fragment ion mass spectra was accomplished with a recently introduced comparative sequencing algorithm (COMPAS). The applied version of COMPAS was specifically optimized for the interpretation of information‐rich spectra obtained on the QqTOF. Sequences of oligodeoxynucleotides as large as 26‐mers were correctly verified in >94% of cases (182 of 192 spectra acquired). Fragment ion mass spectra of larger oligonucleotides were not specific enough for sequencing. Because of the occurrence of extensive internal fragmentation causing low sequence coverage paired with a high probability of assigning fragment ions to wrong sequences, tandem mass spectra obtained from oligonucleotides consisting of 30 and more nucleotides could not be used for sequence verification neither manually nor with COMPAS. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 401–409, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Sequence identity of the n-1 product of a synthetic oligonucleotide.

After synthesis and purification of an oligonucleotide, the final product usually contains a low level of n-1 congeneric species. We have sequenced the n-1 population of a 25mer phosphodiester oligonucleotide. The n-1 band was cut from the gel and eluted. Oligonucleotides were tailed with dA and annealed to a dT-tailed plasmid. The recombinant plasmid was ligated and used to transform competent bacteria. Our results show that the n-1 population was heterogeneous. The frequency of truncated nucleotides at the 3'-end was much higher than at the 5'-end of the oligomer. No truncated nucleotides were found in the last four nucleotides at the 5'-end. Our results also show that the chain of oligonucleotides can grow on unreacted sites of a controlled-pore glass support.     

Synthesis of DNA-oligonucleotides damaged by arylamine-modified 2'-deoxyguanosine

C8-Arylamine-dG adducts bearing a labile N-formamidine group at the exocyclic amino function were converted into their corresponding 5'-O-DMTr-3'-O-phosphoramidite-C8-arylamine-dG derivatives. These compounds were used for the automated synthesis of site-specifically modified oligonucleotides. These oligonucleotides were characterized by ESI-MS and enzymatic digestion and studied for their CD properties and Tm values.     

Observation and elimination of N-acetylation of oligonucleotides prepared using fast-deprotecting phosphoramidites and ultra-mild deprotection

Commercially available 'fast-deprotecting' phosphoramidites are useful for synthesizing oligonucleotides containing alkali-sensitive nucleotides. However, N-acetylated oligonucleotides were observed during solid-phase synthesis using 'fast-deprotecting' phosphoramidites in conjunction with K2CO3/MeOH ('ultra-mild') deprotection. Transamidation was localized at deoxyguanosine, which is protected as its isopropylphenoxyacetyl amide. Substitution of trimethylacetic anhydride for acetic anhydride and appropriate modification of the automated synthesis cycles eliminated this problem.     

Synthesis of oligodeoxyribonucleotides containing oleylamine moieties]

A method for directional introduction of oleylamine residues to any position of oligodeoxyribonucleotides during their automated synthesis was developed. The presence of oleylamine residues in 3'- or 5'-terminal nucleotides was shown to have no effect on the thermodynamic stability of DNA duplexes formed by such oligonucleotides and the complementary sequences. The rate of the snake venom phosphodiesterase hydrolysis of oligonucleotides containing oleylamine residues in the 3'-terminal units was shown to be markedly lower than that of natural oligonucleotides.     

Quantitation of fourteen urinary alpha-amino acids using isobutane gas chromatography chemical ionization mass spectrometry with 13C amino acids as internal standards

Isobutane chemical ionization gas chromatography mass spectometry of the N-trifluoroacetyl-carboxy-n-butyl ester derivatives of amino acids, using a commercial per-13C-amino acid mixture as internal standards, provided a sensitive and specific method for quantitative analysis of fourteen urinary alpha-amino acids. A computer controlled quadrupole mass spectrometer was used in a selected ion monitoring mode to record the ion current due to the protonated molecular ions of each alpha-amino acid/13C analogue pair. BASIC programmes located peak maxima, and using previously established standard curves, calculated the amino acid content on the bases of both peak height and peak area ratios. Duplicate amino acid analyses are possible on 5 microliter of urine. Instrumental analysis required 25 minutes, automated data processing 10 minutes, and sample preparation 2 hours. Detection limits approached 1 ng with a typical mean standard deviation of 2% for the instrumental analysis.     

Mass spectrometry of oligonucleotides

Expanding research in the field of modified oligonucleotides demands suitable analytical tools for size and purity verification of known compounds and accurate structure elucidation of unknowns. There is a need for characterization of the types and sites of modifications in oligonucleotides and to identify and sequence selected candidates originating from synthesis. The potential of electrospray tandem mass spectrometry (ESI-MS/MS) for structural characterization and sequencing of oligonucleotides is demonstrated. The fundamental behavior of DNA, RNA, and selected modified oligonucleotides in gas-phase is shown. Since gas-phase dissociation does not demand specific structural prerequisites, the method bears a great potential for rapid and most accurate characterization of modified oligonucleotides, e.g. from combinatorial libraries.     

Synthesis of Oligodeoxyribonucleotides Containing Oleylamine Moieties

A method for directional introduction of oleylamine residues to any position of oligodeoxyribonucleotides during their automated synthesis was developed. The presence of oleylamine residues in 3"- or 5"-terminal nucleotides was shown to have no effect on the thermodynamic stability of DNA duplexes formed by such oligonucleotides and the complementary sequences. The rate of hydrolysis of oligonucleotides containing oleylamine residues in the 3"-terminal units by the snake venom phosphodiesterase was shown to be markedly lower than that of natural oligonucleotides.     

Rous sarcoma virus Gag protein-oligonucleotide interaction suggests a critical role for protein dimer formation in assembly

The structural protein Gag is the only viral product required for retrovirus assembly. Purified Gag proteins or fragments of Gag are able in vitro to spontaneously form particles resembling immature virions, but this process requires nucleic acid, as well as the nucleocapsid domain of Gag. To examine the role of nucleic acid in the assembly in vitro, we used a purified, slightly truncated version of the Rous sarcoma virus Gag protein, Delta MBD Delta PR, and DNA oligonucleotides composed of the simple repeating sequence GT. Apparent binding constants were determined for oligonucleotides of different lengths, and from these values the binding site size of the protein on the DNA was calculated. The ability of the oligonucleotides to promote assembly in vitro was assessed with a quantitative assay based on electron microscopy. We found that excess zinc or magnesium ion inhibited the formation of virus-like particles without interfering with protein-DNA binding, implying that interaction with nucleic acid is necessary but not sufficient for assembly in vitro. The binding site size of the Delta MBD Delta PR protein, purified in the presence of EDTA to remove zinc ions at the two cysteine-histidine motifs, was estimated to be 11 nucleotides (nt). This value decreased to 8 nt when the protein was purified in the presence of low concentrations of zinc ions. The minimum length of DNA oligonucleotide that promoted efficient assembly in vitro was 22 nt for the zinc-free form of the protein and 16 nt for the zinc-bound form. To account for this striking 1:2 ratio between binding site size and oligonucleotide length requirement, we propose a model in which the role of nucleic acid in assembly is to promote formation of a species of Gag dimer, which itself is a critical intermediate in the polymerizaton of Gag to form the protein shell of the immature virion.     

Fluorescent labelling of sequencing primers for automated oligonucleotide synthesis.

A fluorescein derivative is described which can be used as a normal phosphoramidite in oligonucleotide synthesis, giving high yields of fluorescein labelled sequencing primers. The labelled primers were used in automated DNA-sequence analysis without modification of existing protocols, the computer-processed sequences being reproducibly readable up to 400 bases. The procedure described makes the fluorescent labelling of oligonucleotides much easier, and the time of labelling can be significantly reduced. It speeds up the "primer walking" approach of automated DNA sequencing.     

A rapid method for detecting specific amplified PCR fragments in microtiter plates.

A simple method is presented to circumvent laborious and time consuming electrophoretic separations of specific PCR amplification products. Specific target DNA is amplified using nucleotides labelled with DIG-dUTP or biotin-dCTP. The labelled PCR products are separated from unincorporated nucleotides or oligonucleotides by using a positively charged DEAE cellulose matrix. Amplification products are visualized directly in the matrix using immunoenzymatic methods or streptavidin-conjugated enzymes. The detection process can be carried out within 2 h, allows the processing of large sample sizes and can potentially be automated.     

Rapid Synthesis of a Long Double-Stranded Oligonucleotide from a Single-Stranded Nucleotide Using Magnetic Beads and an Oligo Library

Chemical synthesis of oligonucleotides is a widely used tool in the field of biochemistry. Several methods for gene synthesis have been introduced in the growing area of genomics. In this paper, a novel method of constructing dsDNA is proposed. Short (28-mer) oligo fragments from a library were assembled through successive annealing and ligation processes, followed by PCR. First, two oligo fragments annealed to form a dsDNA molecule. The double-stranded oligo was immobilized onto magnetic beads (solid support) via streptavidin-biotin binding. Next, single-stranded oligo fragments were added successively through ligation to form the complete DNA molecule. The synthesized DNA was amplified through PCR and gel electrophoresis was used to characterize the product. Sanger sequencing showed that more than 97% of the nucleotides matched the expected sequence. Extending the length of the DNA molecule by adding single-stranded oligonucleotides from a basis set (library) via ligation enables a more convenient and rapid mechanism for the design and synthesis of oligonucleotides on the go. Coupled with an automated dispensing system and libraries of short oligo fragments, this novel DNA synthesis method would offer an efficient and cost-effective method for producing dsDNA.     

Improved Polymerase Chain Reaction-restriction Fragment Length Polymorphism Genotyping of Toxic Pufferfish by Liquid Chromatography/Mass Spectrometry

An improved version of a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method for genotyping toxic pufferfish species by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) is described. DNA extraction is carried out using a silica membrane-based DNA extraction kit. After the PCR amplification using a detergent-free PCR buffer, restriction enzymes are added to the solution without purifying the reaction solution. A reverse-phase silica monolith column and a Fourier transform high resolution mass spectrometer having a modified Kingdon trap analyzer are employed for separation and detection, respectively. The mobile phase, consisting of 400 mM 1,1,1,3,3,3-hexafluoro-2-propanol, 15 mM triethylamine (pH 7.9) and methanol, is delivered at a flow rate of 0.4 ml/min. The cycle time for LC/ESI-MS analysis is 8 min including equilibration of the column. Deconvolution software having an isotope distribution model of the oligonucleotide is used to calculate the corresponding monoisotopic mass from the mass spectrum. For analysis of oligonucleotides (range 26-79 nucleotides), mass accuracy was 0.62 ± 0.74 ppm (n = 280) and excellent accuracy and precision were sustained for 180 hr without use of a lock mass standard.     

Collisionally activated dissociation and infrared multiphoton dissociation of oligonucleotides in a quadrupole ion trap

Infrared multiphoton dissociation (IRMPD) of deprotonated and protonated oligonucleotides ranging from 5 to 40 residues has been performed in a quadrupole ion trap mass spectrometer at normal operating pressure and temperature. Only moderate exposure times and laser powers were required to achieve efficient dissociation. In general, IRMPD and collisionally activated dissociation (CAD) produce comparable sequencing information, indicating that IRMPD is a viable alternative to CAD for oligonucleotide analysis in the quadrupole ion trap. Two major characteristics distinguish CAD and IRMPD spectra for a given parent ion. First, structurally uninformative M-B ions that dominate CAD spectra are generally only low-intensity species in IRMPD spectra because nonresonant activation causes these species to dissociate to backbone cleavage products. Second, phosphate and nucleobase ions can be observed directly in IRMPD experiments because the low-mass cutoff can be set to trap small fragment ions. For this reason IRMPD can sometimes facilitate analysis of sequences containing modified bases.     

Electrospray ionisation-cleavable tandem nucleic acid mass tag-peptide nucleic acid conjugates: synthesis and applications to quantitative genomic analysis using electrospray ionisation-MS/MS

The synthesis and characterization of isotopomer tandem nucleic acid mass tag-peptide nucleic acid (TNT-PNA) conjugates is described along with their use as electrospray ionisation-cleavable (ESI-Cleavable) hybridization probes for the detection and quantification of target DNA sequences by electrospray ionisation tandem mass spectrometry (ESI-MS/MS). ESI-cleavable peptide TNT isotopomers were introduced into PNA oligonucleotide sequences in a total synthesis approach. These conjugates were evaluated as hybridization probes for the detection and quantification of immobilized synthetic target DNAs using ESI-MS/MS. In these experiments, the PNA portion of the conjugate acts as a hybridization probe, whereas the peptide TNT is released in a collision-based process during the ionization of the probe conjugate in the electrospray ion source. The cleaved TNT acts as a uniquely resolvable marker to identify and quantify a unique target DNA sequence. The method should be applicable to a wide variety of assays requiring highly multiplexed, quantitative DNA/RNA analysis, including gene expression monitoring, genetic profiling and the detection of pathogens.     

Characterization of synthetic oligonucleotides containing biologically important modified bases by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Oligonucleotides containing modified bases are commonly used for biochemical and biophysical studies to assess the impact of specific types of chemical damage on DNA structure and function. In contrast to the synthesis of oligonucleotides with normal DNA bases, oligonucleotide synthesis with modified bases often requires modified synthetic or deprotection conditions. Furthermore, several modified bases of biological interest are prone to further damage during synthesis and oligonucleotide isolation. In this article, we describe the application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to the characterization of a series of modified synthetic oligonucleotides. The potential for and limits in obtaining high mass accuracy for confirming oligonucleotide composition are discussed. Examination of the isotope cluster is also proposed as a method for confirming oligonucleotide elemental composition. MALDI-TOF-MS analysis of the unpurified reaction mixture can be used to confirm synthetic sequence and to reveal potential problems during synthesis. Analysis during and after purification can yield important information on depurination and base oxidation. It can also reveal unexpected problems that can occur with nonstandard synthesis, deprotection, or purification strategies. Proper characterization of modified oligonucleotides is essential for the correct interpretation of experiments performed with these substrates, and MALDI-TOF-MS analysis provides a simple yet extensive method of characterization that can be used at multiple stages of oligonucleotide production and use.     

Trityl mass-tags for encoding in combinatorial oligonucleotide synthesis.

Combinatorial libraries of oligonucleotides on beads were synthesised by a split-and-mix strategy using 5'-DMTr- or 5'-Fmoc- nucleoside phosphoramidites. Trityl moieties with different masses were used to encode for the bases coupled at each step in the synthesis of oligonucleotides selected by hybridisation from the libraries. Tags orthogonal to the nucleotides added were produced by coupling amines of different MW to an activated carboxyl group(s) on the trityl moiety. Tags can be released from the support by laser irradiation and measured directly by TOF without matrix. Alternatively, they may be released by an acidic treatment and then analysed by (MA)LDI-TOF.     

Sequence and location of large RNase T1 oligonucleotides in bacteriophage Qbeta RNA.

Twenty-nine oligonucleotides, 11 to 26 nucleotides in length, arising by complete RNase T1 digestion of bacteriophage Qbeta RNA and isolated by two-dimensional polyacrylamide gel electrophoresis, were sequenced. Their location within the genome was established with two methods. (a) In vitro synthesis of Qbeta RNA plus strands was started synchronously, using minus strands as template and nucleoside [alpha-32P]triphosphates as substrate; after various times, the reaction was stopped and the length of the products formed was correlated with their content of T1 oligonucleotides. (b) Qbeta [32P]RNA was elongated with poly(A) using terminal riboadenylate transferase; after mild treatment with alkali the fragments were fractionated by size and the poly(A)-containing molecules of each size class were isolated by chromatography on poly(U)-Sephadex and assayed for T1 oligonucleotides. The oligonucleotides in the 5' region were localized more precisely with method a, those near the 3' end with method b; in the middle region, the results of the two sets of analyses confirmed each other. The use of these oligonucleotides in the sequence determination of Qbeta RNA is discussed.     

Characterisation of glucosinolates using electrospray ion trap and electrospray quadrupole time-of-flight mass spectrometry

Twelve naturally occurring glucosinolates displaying alkenyl, hydroxylated, methylsulphinyl, aromatic and indole side chains were investigated by both negative and positive ion electrospray ionisation-tandem mass spectrometry (ESI-MS/MS). In order to resolve the MS/MS spectra obtained from the anion and cation molecular ions of glucosinolates, the different fragments were investigated by MSn experiments using an ion trap spectrometer. The MS3 spectra obtained permitted possible fragmentation schemes to be proposed. These were supported by accurate mass measurements of some characteristic diagnostic ions with the help of a quadrupole time-of-flight instrument. The negative ion ESI-MS/MS behaviour of the different glucosinolates investigated in this study confirmed previously described patterns and revealed new interesting structural informative fragments. Some are common to all the glucosinolates and others are highly specific for a type of variable side chain. The positive ion ESI-MS/MS fragments obtained from the [MNa+Na]+ or [MK+K]+ molecular ions did not provide complementary specific diagnostic ions. Nevertheless, when compared with the negative ion mode, the daughter ions appeared more homogenous and with a better relative abundance for all of the 12 compounds studied. Moreover, the positive ion mode appeared to be more efficient than the negative mode for the study of methoxylated glucosinolates and should be useful to detect the glucosinolates present as organic salts in crude plant extracts.