Tandem mass spectrometric sequence characterization of synthetic thymidine-rich oligonucleotides

Tandem mass spectrometry (MS/MS) can provide direct and accurate sequence characterization of synthetic oligonucleotide drugs, including modified oligonucleotides. Multiple factors can affect oligonucleotide MS/MS sequencing, including the intrinsic properties of oligonucleotides (i.e., nucleotide composition and structural modifications) and instrument parameters associated with the ion activation for fragmentation. In this study, MS/MS sequencing of a thymidine (T)-rich and phosphorothioate (PS)-modified DNA oligonucleotide was investigated using two fragmentation techniques: trap-type collision-induced dissociation (“CID”) and beam-type CID also termed as higher-energy collisional dissociation (“HCD”), preceded by a hydrophilic interaction liquid chromatography (HILIC) separation. A low to moderate charge state (−4), which predominated under the optimized HILIC-MS conditions, was selected as the precursor ion for MS/MS analysis. Comparison of the two distinctive ion activation mechanisms on the same precursor demonstrated that HCD was superior to CID in promoting higher sequence coverage and analytical sensitivity in sequence elucidation of T-rich DNA oligonucleotides. Specifically, HCD provided more sequence-defining fragments with higher fragment intensities than CID. Furthermore, the direct comparison between unmodified and PS-modified DNA oligonucleotides demonstrated a loss of MS/MS fragmentation efficiency by PS modification in both CID and HCD approaches, and a resultant reduction in sequence coverage. The deficiency in PS DNA sequence coverage observed with single collision energy HCD, however, was partially recovered by applying HCD with multiple collision energies. Collectively, this work demonstrated that HCD is advantageous to MS/MS sequencing of T-rich PS-modified DNA oligonucleotides.     

Sequence verification of oligonucleotides containing multiple arylamine modifications by enzymatic digestion and liquid chromatography mass spectrometry (LC/MS)

An analytical method for the structure differentiation of arylamine modified oligonucleotides (ODNs) using on-line LC/MS analysis of raw exonuclease digests is described. Six different dodeca ODNs derived from the reaction of N-acetoxy-N-(trifluoroacetyl)-2-aminofluorene with the dodeca oligonucleotide 5'-CTCGGCGCCATC-3' are isolated and sequenced with this LC/MS method using 3'- and 5'-exonucleases. When the three products modified by a single aminofluorene (AF) are subjected to 3'-exonuclease digestion, the exonuclease will cleave a modified nucleotide but when di-AF modified ODNs are analyzed the 3'-exonuclease ceases to cleave nucleotides when the first modification is exposed at the 3'-terminus. Small abundances of ODN fragments formed by the cleavage of an AF-modified nucleotide were observed when two of the three di-AF modified ODNs were subjected to 5'-exonuclease digestion. The results of the 5'-exonuclease studies of the three di-AF modified ODNs suggest that as the number of unmodified bases between two modifications in an ODN sequence increases, the easier it becomes to sequence beyond the modification closest to the 5'-terminus. The results of this study indicate that the LC/MS method described here would be useful in sequencing ODNs modified by multiple arylamines to be used as templates for site-specific mutagenesis studies.     

Electrospray ionization tandem mass spectrometry of biphenyl-modified oligo(deoxy)ribonucleotides

Antisense oligonucleotides and aptamers are important candidates for future therapeutic applications. Different structural modifications are introduced into oligonucleotides to obtain high affinity and binding specificity. Sequence elucidation of oligonucleotides incorporating a wide variety of modifications presents an analytical challenge, as the standard protocols cannot be applied. Mass spectrometry has the potential to solve complex structural problems. However, a better understanding of the fundamental aspects of gas-phase dissociation of modified DNA and RNA is needed. In this work the influence of specific chemical modifications on backbone dissociation is pointed out. Biphenyl-modified oligo(deoxy)ribonucleotides, which incorporate C-glycosidic bound abasic nucleobase substitutes, were subjected to collision-induced dissociation in an electrospray tandem mass spectrometer, with the goal to investigate the role of nucleobase loss on backbone dissociation. DNA bearing biphenyl nucleobase substitutes show abundant [a-B]- and w-ions generated by cleavage of the 3'-C-O bonds, except for the phosphodiester groups adjacent to the biphenyl modifications. At these positions no dissociation was observed, demonstrating the dependence of DNA backbone dissociation on nucleobase loss. Also, no evidence for a base loss independent mechanism responsible for formation of w-ions was found. RNA incorporating biphenyl nucleobase substitutes fragment into c- and y-ions resulting from cleavage of the 5'-P-O bond. Adjacent to the biphenyl modifications no altered dissociation behavior was found. This leads to the conclusion that dissociation of RNA is independent of the 1'-modification and, therefore, independent of nucleobase loss.     

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.     

Designed diblock oligonucleotide for the synthesis of spatially isolated and highly hybridizable functionalization of DNA-gold nanoparticle nanoconjugates

Conjugates of DNA and gold nanoparticles (AuNPs) typically exploit the strong Au-S chemistry to self-assemble thiolated oligonucleotides at AuNPs. However, it remains challenging to precisely control the orientation and conformation of surface-tethered oligonucleotides and finely tune the hybridization ability. We herein report a novel strategy for spatially controlled functionalization of AuNPs with designed diblock oligonucleotides that are free of modifications. We have demonstrated that poly adenine (polyA) can serve as an effective anchoring block for preferential binding with the AuNP surface, and the appended recognition block adopts an upright conformation that favors DNA hybridization. The lateral spacing and surface density of DNA on AuNPs can also be systematically modulated by adjusting the length of the polyA block. Significantly, this diblock oligonucleotide strategy results in DNA-AuNPs nanoconjugates with high and tunable hybridization ability, which form the basis of a rapid plasmonic DNA sensor.     

De novo sequencing of highly modified therapeutic oligonucleotides by hydrophobic tag sequencing coupled with LC–MS

Correct sequences are prerequisite for quality control of therapeutic oligonucleotides. However, there is no definitive method available for determining sequences of highly modified therapeutic RNAs, and thereby, most of the oligonucleotides have been used clinically without direct sequence determination. In this study, we developed a novel sequencing method called ‘hydrophobic tag sequencing’. Highly modified oligonucleotides are sequenced by partially digesting oligonucleotides conjugated with a 5′‐hydrophobic tag, followed by liquid chromatography–mass spectrometry analysis. 5′‐Hydrophobic tag‐printed fragments (5′‐tag degradates) can be separated in order of their molecular masses from tag‐free oligonucleotides by reversed‐phase liquid chromatography. As models for the sequencing, the anti‐VEGF aptamer (Macugen) and the highly modified 38‐mer RNA sequences were analyzed under blind conditions. Most nucleotides were identified from the molecular weight of hydrophobic 5′‐tag degradates calculated from monoisotopic mass in simple full mass data. When monoisotopic mass could not be assigned, the nucleotide was estimated using the molecular weight of the most abundant mass. The sequences of Macugen and 38‐mer RNA perfectly matched the theoretical sequences. The hydrophobic tag sequencing worked well to obtain simple full mass data, resulting in accurate and clear sequencing. The present study provides for the first time a de novo sequencing technology for highly modified RNAs and contributes to quality control of therapeutic oligonucleotides. Copyright © 2016 John Wiley & Sons, Ltd.     

Synthesis and properties of novel l-isonucleoside modified oligonucleotides and siRNAs

Antisense oligonucleotides and siRNAs are potential therapeutic agents and their chemical modifications play an important role to improve the properties and activities of oligonucleotides. Isonucleoside is a type of nucleoside analogue, in which the nucleobase is moved from C-1 to other positions of ribose. In this report, a novel isonucleoside containing a 5'-CH(2)-extended chain at the sugar moiety was synthesized, thus isoadenosine and isothymidine were incorporated into a DNA single strand and siRNA. It was found that isonucleoside modified oligonucleotides can form stable double helical structures with their complementary DNA and RNA and the stability towards nuclease and ability to activate RNase H are more promising compared with the unmodified, natural analogues. In siRNA, passenger strand modified with isonucleoside () at 3' or 5' terminal can retain the silencing activity and minimize the passenger strand specific off-target effect.     

Template-Directed Copying of RNA by Non-enzymatic Ligation

The non-enzymatic replication of the primordial genetic material is thought to have enabled the evolution of early forms of RNA-based life. However, the replication of oligonucleotides long enough to encode catalytic functions is problematic due to the low efficiency of template copying with mononucleotides. We show that template-directed ligation can assemble long RNAs from shorter oligonucleotides, which would be easier to replicate. The rate of ligation can be greatly enhanced by employing a 3′-amino group at the 3′-end of each oligonucleotide, in combination with an N-alkyl imidazole organocatalyst. These modifications enable the copying of RNA templates by the multistep ligation of tetranucleotide building blocks, as well as the assembly of long oligonucleotides using short splint oligonucleotides. We also demonstrate the formation of long oligonucleotides inside model prebiotic vesicles, which suggests a potential route to the assembly of artificial cells capable of evolution.     

An algorithm for interpretation of low-energy collision-induced dissociation product ion spectra for de novo sequencing of peptides

An algorithm for interpretation of product ion spectra of peptides generated from ion trap mass spectrometry is developed for de novo amino acid sequencing of peptides for the purpose of protein identification. It is based on a multi-pass analysis of product ion data using a rigorous data extraction and sequence interpretation protocol in the initial pass. The extraction/interpretation algorithm becomes more relaxed in subsequent passes, considering more of the fragment ions, and potentially more sequence candidates. The possible peptide sequences generated by the algorithm are scored according to those sequences which best explain the fragment ion spectrum. These sequences are searched against a protein database using a BLAST search engine to find likely protein candidates. The method is also suitable for locating and determining protein modifications, and can be applied to de novo interpretation of peptide fragment ions in the tandem mass (MS/MS) spectrum produced from a mixture of two peptides having similar nominal mass, but different sequences. Using a known protein, bovine serum albumin, as an example, it is illustrated that this method is rapid and efficient for MS/MS spectral interpretation. This method combined with BLAST programs is then applied to search homologies and to generate information on post-translational modifications of an unknown protein isolated from shark cartilage that does not have a complete genome or proteome database.     

Template‐Directed Copying of RNA by Non‐enzymatic Ligation

The non‐enzymatic replication of the primordial genetic material is thought to have enabled the evolution of early forms of RNA‐based life. However, the replication of oligonucleotides long enough to encode catalytic functions is problematic due to the low efficiency of template copying with mononucleotides. We show that template‐directed ligation can assemble long RNAs from shorter oligonucleotides, which would be easier to replicate. The rate of ligation can be greatly enhanced by employing a 3′‐amino group at the 3′‐end of each oligonucleotide, in combination with an N‐alkyl imidazole organocatalyst. These modifications enable the copying of RNA templates by the multistep ligation of tetranucleotide building blocks, as well as the assembly of long oligonucleotides using short splint oligonucleotides. We also demonstrate the formation of long oligonucleotides inside model prebiotic vesicles, which suggests a potential route to the assembly of artificial cells capable of evolution.     

Simulating the Monty Hall problem in a DNA sequencing machine

The Monty Hall problem is a decision problem with an answer that is surprisingly counter-intuitive yet provably correct. Here we simulate and prove this decision in a high-throughput DNA sequencing machine, using a simple encoding. All possible scenarios are represented by DNA oligonucleotides, and gameplay decisions are implemented by sequencing these oligonucleotides from specific positions, with a single run simulating more than 12,000,000 independent games. This work highlights high-throughput DNA sequencing as a new tool that could extend existing capabilities and enable new encoding schemes for problems in DNA computing.     

Positive ion formation in the ultraviolet matrix-assisted laser desorption / ionization analysis of oligonucleotides by using 2,5-dihydroxybenzoic acid

The development of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry and its demonstrated performance with large proteins has generated substantial interest in utilizing this technique as an alternative to gel electrophoresis for DNA sequence analysis. However, a lack of understanding of the desorption and ionization processes has greatly hampered advances in this field. This article explores the formation of positively charged oligonucleotides in UV (355-nm) MALDI analysis by using the matrix 2,5-dihydroxybenzoic acid. Whereas substantial fragmentation is observed in the positive-ion mode by using the short oligomer d(TAGGT), no fragmentation is evident in the negative-ion mode under identical conditions. The fragmentation products are consistent with a previously published model in which base protonation initiates base loss, which leads to subsequent cleavage of the phosphodiester backbone. Several polydeoxythymidilic acids containing modified nucleosides were used to investigate positive-ion formation. The results support the hypothesis that positive ions are formed by protonation of the nucleobases. Because base protonation initiates base loss, fragmentation is intrinsic to positive-ion formation in the MALDI analysis of oligonucleotides. This result explains the dramatic difference in fragmentation observed in positive-ion compared to negative-ion UV-MALDI mass spectra of oligonucleotides.     

DETECTING LOW-LEVEL SYNTHESIS IMPURITIES IN MODIFIED PHOSPHOROTHIOATE OLIGONUCLEOTIDES USING LIQUID CHROMATOGRAPHY - HIGH RESOLUTION MASS SPECTROMETRY

An LC-MS method based on the use of high resolution Fourier transform ion cyclotron resonance mass spectrometry (FTIRCMS) for profiling oligonucleotides synthesis impurities is described.Oligonucleotide phosphorothioatediesters (phosphorothioate oligonucleotides), in which one of the non-bridging oxygen atoms at each phosphorus center is replaced by a sulfur atom, are now one of the most popular oligonucleotide modifications due to their ease of chemical synthesis and advantageous pharmacokinetic properties. Despite significant progress in the solid-phase oligomerization chemistry used in the manufacturing of these oligonucleotides, multiple classes of low-level impurities always accompany synthetic oligonucleotides. Liquid chromatography-mass spectrometry has emerged as a powerful technique for the identification of these synthesis impurities. However, impurity profiling, where the entire complement of low-level synthetic impurities is identified in a single analysis, is more challenging. Here we present an LC-MS method based the use of high resolution-mass spectrometry, specifically Fourier transform ion cyclotron resonance mass spectrometry (FTIRCMS or FTMS). The optimal LC-FTMS conditions, including the stationary phase and mobile phases for the separation and identification of phosphorothioate oligonucleotides, were found. The characteristics of FTMS enable charge state determination from single m/z values of low-level impurities. Charge state information then enables more accurate modeling of the detected isotopic distribution for identification of the chemical composition of the detected impurity. Using this approach, a number of phosphorothioate impurities can be detected by LC-FTMS including failure sequences carrying 3'-terminal phosphate monoester and 3'-terminal phosphorothioate monoester, incomplete backbone sulfurization and desulfurization products, high molecular weight impurities, and chloral, isobutyryl, and N(3) (2-cyanoethyl) adducts of the full length product. When compared with low resolution LC-MS, ~60% more impurities can be identified when charge state and isotopic distribution information is available and used for impurity profiling.     

Biopolymer sequencing using a triple quadrupole mass spectrometer in the ESI nozzle-skimmer/precursor ion MS/MS mode

A variety of model biopolymers, including oligonucleotides, oligosaccharides and a synthetic pharmaceutical agent, were sequenced using a triple quadrupole mass spectrometer equipped with an electrospray source and operated in a scan mode referred to as pseudo-MS3. This scan mode consists of three steps: (1) in-source collision-induced dissociation (CID) in the nozzle-skimmer (NS) region, (2) scanning of the fragment ions into the collision cell for further CID, and (3) passing of the secondary fragment ions through the final mass filter at a preselected mass, generally corresponding to the mass of a terminal sequence ion for the biopolymer. The mass spectra are recorded in the precursor ion MS/MS mode where ion selection and detection occur at the third stage of the triple quadrupole but the scan function is determined by the first stage. The advantages and limitations in using this pseudo-MS3 NS/precursor ion MS/MS scan mode for biopolymer sequencing are discussed.     

Formation of diagnostic product ions from cyanobacterial cyclic peptides by the two-bond fission mechanism using ion trap liquid chromatography/multi-stage mass spectrometry

Product ions obtained by tandem mass spectrometry (MS/MS) are quite effective for the amino acid sequencing of linear peptides. However, in the case of cyclic peptides, the fragmentation pattern is complicated because the cleavages occur randomly and product ions are generated as a(n), b(n), c(n), x(n), y(n) and z(n) series ions; therefore, the authors have never obtained sufficient sequence information. In order to overcome this problem, we applied ion trap liquid chromatography/multi-stage mass spectrometry (LC/MS(n)) and characterized the product ions obtained from anabaenopeptins and aeruginopeptins as the cyclic peptides. For the anabaenopeptins, MS(2) analysis did not provide sufficient sequence information on the cyclic structure, and MS(3) analysis was applied to sequence the constituent amino acids. Diagnostic product ions were obtained by the MS(3) analysis and were quite effective for obtaining the sequence information of the constituent amino acids. MS(2) analysis was, however, sufficient to obtain the sequence information of the aeruginopeptins. In both cases, the resulting product ions obtained from the cyclic structures were formed by the two-bond fission mechanism of the precursor ion, in which an initial fission of the cyclic structure to a linear one and subsequent fission(s) at the peptide bonds are included. The fragmentations were similar for the structurally related compounds, indicating that the cleavages occurred at definite peptide bonds. In addition, the resulting product ions are generated as b(n) series ions and the mass difference facilitates the amino acid sequencing. Thus, ion trap LC/MS(n) provides sequence information, and the resulting product ions are reproducible among the structurally related compounds and reliable for the sequencing of the constituent amino acids of the cyclic structure.     

Recent developments in DNA sequencing by capillary and microdevice electrophoresis

The present review covers papers published in the years 1997 and 1998 on DNA sequencing by capillary and microdevice electrophoresis. The article does not include other electrophoretic DNA applications such as analysis of oligonucleotides, genotyping, and mutational analysis. Capillary gel electrophoresis (CGE) is starting to become a viable competitor to slab gel electrophoresis for DNA sequencing. Commercially available multicapillary array sequencers are now entering sequencing facilities which to date have totally relied on traditional slab gel technology. CGE research on DNA sequencing therefore becomes increasingly concerned with the critical task of fine-tuning the operational parameters to create robust sequencing systems. Electrophoretic microdevices are being considered the next technological step in DNA sequencing by electrophoresis.     

Insertion of Chemical Handles into the Backbone of DNA during Solid-Phase Synthesis by Oxidative Coupling of Amines to Phosphites

Conjugation of molecules or proteins to oligonucleotides can improve their functional and therapeutic capacity. However, such modifications are often limited to the 5′ and 3′ end of oligonucleotides. Herein, we report the development of an inexpensive and simple method that allows for the insertion of chemical handles into the backbone of oligonucleotides. This method is compatible with standardized automated solid-phase oligonucleotide synthesis, and relies on formation of phosphoramidates. A unique phosphoramidite is incorporated into a growing oligonucleotide, and oxidized to the desired phosphoramidate using iodine and an amine of choice. Azides, alkynes, amines, and alkanes have been linked to oligonucleotides via internally positioned phosphoramidates with oxidative coupling yields above 80 %. We show the design of phosphoramidates from secondary amines that specifically hydrolyze to the phosphate only at decreased pH. Finally, we show the synthesis of an antibody-DNA conjugate, where the oligonucleotide can be selectively released in a pH 5.5 buffer.     

Effects of transition metal ion coordination on the collision-induced dissociation of polyalanines

Transition metal-polyalanine complexes were analyzed in a high-capacity quadrupole ion trap after electrospray ionization. Polyalanines have no polar amino acid side chains to coordinate metal ions, thus allowing the effects metal ion interaction with the peptide backbone to be explored. Positive mode mass spectra produced from peptides mixed with salts of the first row transition metals Cr(III), Fe(II), Fe(III), Co(II), Ni(II), Cu(I), and Cu(II) yield singly and doubly charged metallated ions. These precursor ions undergo collision-induced dissociation (CID) to give almost exclusively metallated N-terminal product ions whose types and relative abundances depend on the identity of the transition metal. For example, Cr(III)-cationized peptides yield CID spectra that are complex and have several neutral losses, whereas Fe(III)-cationized peptides dissociate to give intense non-metallated products. The addition of Cu(II) shows the most promise for sequencing. Spectra obtained from the CID of singly and doubly charged Cu-heptaalanine ions, [M + Cu - H](+) and [M + Cu](2+) , are complimentary and together provide cleavage at every residue and no neutral losses. (This contrasts with [M + H](+) of heptaalanine, where CID does not provide backbone ions to sequence the first three residues.) Transition metal cationization produces abundant metallated a-ions by CID, unlike protonated peptides that produce primarily b- and y-ions. The prominence of metallated a-ions is interesting because they do not always form from b-ions. Tandem mass spectrometry on metallated (Met = metal) a- and b-ions indicate that [b(n) + Met - H](2+) lose CO to form [a(n) + Met - H](2+), mimicking protonated structures. In contrast, [a(n) + Met - H](2+) eliminate an amino acid residue to form [a(n-1) + Met - H](2+), which may be useful in sequencing.     

Factors determining the performance of triple quadrupole, quadrupole ion trap and sector field mass spectrometer in electrospray ionization mass spectrometry. 2. Suitability for de novo sequencing

The sequence coverage by fragment ions resulting from collision-induced dissociation in a triple stage quadrupole (TSQ) and a quadrupole ion trap (QIT) mass spectrometer of 10-20-mer oligonucleotides was investigated. While (a-B) and w ion series were the most abundant on both instruments, additional ion series of sequence relevance were preferably formed in the TSQ. Thus, a total number of 83 fragment ions were used to deduce the complete sequence of a 10-mer oligonucleotide of mixed sequence from a tandem mass spectrum recorded on the TSQ. The complete sequence was also encoded in the 28 fragments that were obtained from the QIT under comparable fragmentation conditions. Spectrum complexity increased considerably at the cost of signal-to-noise ratio upon fragmentation of a 20-mer oligonucleotide in the TSQ, whereas spectrum interpretation with longer oligonucleotides was significantly more straightforward in spectra recorded on the QIT. The extent of fragmentation had to be optimized by appropriate setting of collision energy and choice of precursor ion charge state in order to obtain full sequence coverage by fragments for de novo sequencing. Moreover, full sequence information was also dependent on base sequence because of the low tendency of backbone cleavage at thymidines. Tandem mass spectrometry on the QIT yielded redundant information that was successfully utilized to deduce the complete sequence of 20-mer oligonucleotides with high confidence.     

Gas-phase stability of double-stranded oligodeoxynucleotides and their noncovalent complexes with DNA-binding drugs as revealed by collisional activation in an ion trap

The intrinsic (gas-phase) stabilities of duplex, self-complementary oligonucleotides were measured in a relative way by subjecting the duplex precursor ions to increasing amounts of collision energy during the collisional-activated decomposition (CAD) events in an ion-trap mass spectrometer. The results are displayed as a dissociation profile, an s-shaped curve that shows the dependence of the relative abundance of the duplex on the applied collision energy. The total number of charges, the total number of base pairs, and the location of the high proton-affinity bases (i.e., G and C) are the main factors that affect the intrinsic stability of the duplex oligonucleotides. If the charge state is the same, the stability, as measured as a half-wave collision energy, E1/2, correlates well with the total number of H bonds for the duplex. The intrinsic stabilities of noncovalent complexes between duplex oligonucleotide and some DNA-binding drugs were also measured by using the newly developed method. Although duplexes are stabilized in the gas phase when they bind to drug molecules, correlations between gas-phase stabilities and the solution-binding affinities have not yet been obtained. Complexes in which the drug is bound in the minor groove must be joined tightly because they tend to dissociate in the gas phase by breaking covalent bonds of the oligonucleotide to give base loss and small sequence-ion formation. Complexes in which the drug is known to favor intercalation dissociate by breaking weak, noncovalent bonds to form single-stranded oligonucleotides although cleavage of covalent bonds of the oligonucleotide also occurs.