Rapid Sequencing of Oligonucleotides by High-Resolution Mass Spectrometry

Modified oligonucleotides continue to play an important role as antisense compounds that inhibit the expression of genes associated with metabolic disorders, cancer, and infectious diseases. Because the majority of modifications render these molecules refractory to standard enzymatic sequencing techniques, alternative sequencing methods which are fast and reliable are needed. In this work we explore how sugar and backbone modifications affect fragmentation patterns observed from oligonucleotides which are fragmented by infrared multiple photon dissociation in the external reservoir of an electrospray ionization Fourier transform ion cyclotron mass spectrometer. The modifications influence which fragment types (i.e., a n -B versus c n ) dominate and the ease with which the oligonucleotides are fragmented. General observations for confirming the sequence of oligonucleotides are described. A ntisense compounds are modified oligonucleotides that bind messenger RNA and inhibit the expression of genes associated with metabolic disorders, cancer, and infectious diseases. Modified oligonucleotides are necessary to increase the binding of the oligonucleotides to the messenger RNA as well as to minimize the in vivo enzymatic degradation of the oligonucleotides [1]. Typically, modifications are made to the phosphate backbone and to the 2Ј position of the sugar. Fast and reliable methods are needed for sequencing these oligonucleotides since standard enzymatic techniques are not compatible with the majority of these modifications.Mass spectrometry has developed into a powerful tool for studying macromolecules of biological importance, including nucleic acids. Soft ionization techniques such as electrospray ionization [2-4] and matrixassisted laser desorption/ionization [5,6] allow the introduction of large intact biomolecules into the gas phase for mass spectrometric analysis. In addition to accurate molecular weight determination, mass spectrometry in combination with different fragmentation schemes provides information about sequence and/or structure of the biomolecules. Seminal work by McLafferty and coworkers demonstrated the ability to obtain partial sequence information for DNA oligonucleotides up to 108 nucleotides in length using tandem mass spectrometry techniques on a FTICR spectrometer [7]. By combining various fragmentation schemes, it was shown that complete sequence coverage could be obtained for a 50-mer DNA [7][8][9]. Other researchers demonstrated that sequence information and identification of modified bases could be obtained by combining fragmentation schemes with ion trap mass spectrometry [10] and triple quadrupole mass spectrometry [11][12][13].Based on the small amounts of sample required and the unambiguous signatures generated, mass spectrometry is emerging as the preferred platform on which to interrogate and sequence modified oligonucleotides. In order to faithfully sequence oligonucleotides containing desperate chemical modifications, an understanding of how the modifications influence the fragme...

Section: Introductionmentioning

confidence: 99%

Sequence confirmation of modified oligonucleotides using IRMPD in the external ion reservoir of an electrospray ionization fourier transform ion cyclotron mass spectrometer

Sannes‐Lowery

Hofstadler

2003

“…A critical component of many of these experiments is the application of tandem mass spectrometry to probe the fragments produced when DNA ions [34 -36], noncovalent DNA-drug complexes [37,38], or covalent DNA-drug complexes [39 -55] are energized in the gas phase. Collisionally-activated dissociation (CAD) [56], infrared multiphoton dissociation (IRMPD) [57,58], and electron capture dissociation (ECD) [59] have all been demonstrated to yield useful information about oligonucleotides. With respect to covalently bound DNA adducts, such as the ones discussed in this study, determination of both the position and mode of attachment of a drug or ligand to a DNA sequence has been the primary focus of many of these prior reports [39 -55].…”

mentioning

confidence: 99%

Identification of the adduct between a 4-aza-3-ene-1,6-diyne and DNA using electrospray ionization mass spectrometry

Sherman

Pierce

Brodbelt

et al. 2006

The interactions between a novel enediyne [1-methyl-2-(phenylethynyl)-3-(3-phenylprop-2-ynyl)-3H-benzimidazolium] (1) and various cytosine-containing oligonucleotides were studied using electrospray ionization mass spectrometry (ESI-MS) in a flow injection analysis mode useful for small volumes. This enediyne ligand, developed as a potential alternative to the highly cytotoxic natural enediynes, some of which have been successfully used as anti-tumor agents, has previously been shown to interact with DNA through frank strand scission as well as via the formation of adducts that lead to 2=-deoxycytidine-specific cleavage. Through ESI-MS, the structures of these adducts were examined and a sequence dependence of the 2=-deoxycytidine-specific cleavage was noted. Collisionally activated dissociation of the observed adducts confirmed the strength of the interactions between the enediyne and DNA and supports a direct linkage between the enediyne and the cytosine nucleobase, likely the result of a nucleophilic attack of the phenylethynyl group by the cytosine amine. , much effort has been directed towards identifying additional natural enediynes and evaluating their activities [2][3][4][5]. Currently, nearly twenty of these natural enediynes have been identified, including calichemicin ␥ 1 I [6,7], dynemicin [8], and the esperamicins [9,10]. While these enediynes offer extremely high cytotoxic activities, their clinical use as anti-tumor agents has been limited due to their delayed activities and general toxicity [2][3][4][5].To overcome the shortcomings of these natural enediynes, a range of synthetic analogs have been explored [3,11], each with the goal of improved properties for clinical use. While still capable of cleaving DNA like the natural products through cycloaromatization reactions and subsequent hydride abstraction by the diradical intermediate [2-4, 11, 12], these analogs offer potential advantages, such as more controlled activation of the compound in vivo [13][14][15][16][17][18][19][20][21][22][23] or increased selectivity for tumor cells [24 -30].To better evaluate the activities and properties of the many enediyne drug candidates as well as various other classes of drugs being developed, we present a technique for analyzing the interactions between these compounds and DNA using a flow injection analysis (FIA) mode coupled to an electrospray ionization mass spectrometer (ESI-MS). This analysis offers several key advantages over the traditional gel-based electrophoretic techniques. First, the dramatically better resolution of mass spectrometry versus polyacrylamide gel electrophoresis (PAGE) allows differentiation of similarly sized species, enabling the detection of very small molecules adducted to DNA. Second, while gel electrophoresis typically requires micrograms of sample, with our FIA method, a mass spectrum can easily be collected with nanograms, a sensitivity advantage of up to three orders of magnitude. Lastly, FIA-ESI-MS is easily adaptable to high-throughput analyses [31][32][33].In recent y...

“…The starting point of the sequencing strategy relies on the fact that high accurate molecular mass measurements enable the determination of the nucleotide composition of a DNA molecule [11,25,26]. In this context, Russel and coworkers demonstrated that a mass accuracy of better than Ϯ1 ppm, which can be routinely achieved on time-of-flight (TOF) and Fourier transform ion cyclotron resonance (FTICR) instruments, allows the unequivocal assignment of the composition of 20-mers [26].…”

Section: De Novo Sequencing Strategymentioning

confidence: 99%

“…Consequently, MSMS of oligodeoxynucleotides has been successfully applied to characterize or confirm structures [11], to detect chemical modifications [2,10,18], and to identify sequence variations [14, 19 -21]. However, the deduction of sequence information is time-consuming, highly technical, lacking strict objectivity due to reliance on human interpretation, and can be performed only in laboratories with extensive experience in MSMS.…”

mentioning

confidence: 99%

Automated de novo sequencing of nucleic acids by liquid chromatography-tandem mass spectrometry

Oberacher

Mayr

Huber

2004

We present the first global computer-aided sequencing algorithm for the de novo determination of short nucleic acid sequences. The method compares the fragment ion spectra generated by collision-induced dissociation of multiply charged oligodeoxynucleotide-ions to the m/z values predicted employing established fragmentation pathways from a known reference sequence. The closeness of matching between the measured spectrum and the predicted set of fragment ions is characterized by the fitness, which takes into account the difference between measured and predicted m/z values, the intensity of the fragment ions, the number of fragments assigned, and the number of nucleotide positions not covered by fragment ions in the experimental spectrum. Smaller values for the fitness indicate a closer match between the measured spectrum and predicted m/z values. In order to find the sequence most closely matching the experimental spectrum, starting from a given nucleotide composition all possible oligonucleotide sequences are assembled followed by identification of the correct sequence by the lowest fitness value. Using this concept, sequences of 5-to 12-mer oligodeoxynucleotides were successfully de novo determined. High sequence coverage with fragment ions was essential for obtaining unequivocal sequencing results. Moreover, the collision energy was shown to have an impact on the interpretability of tandem mass spectra by the de novo sequencing algorithm. Experiments revealed that the optimal collision energy should be set to a value just sufficient for complete fragmentation of the precursor ion. T he hyphenation of liquid chromatography (LC) to mass spectrometry (MS) is one of the most powerful methods for the characterization of oligonucleotides today [1]. In this context the combination of ion-pair reversed-phase high-performance liquid chromatography and electrospray ionization mass spectrometry (ICEMS) has emerged as a versatile tool for the analysis of single-and double-stranded nucleic acids ranging in size from a few nucleotides (nt) up to several hundred base pairs (bp) [2][3][4]. Although the occurrence of a sequence variation is predictable on the basis of molecular mass measurements [5][6][7], detailed sequencing information can only be obtained from selective decomposition of oligonucleotides by tandem mass spectrometry (MSMS).The principles of gas-phase collision-induced dissociation (CID) reactions of oligonucleotides have been studied extensively [8 -17] and the resulting product ion spectra are predictable on the basis of known fragmentation pathways. Consequently, MSMS of oligodeoxynucleotides has been successfully applied to characterize or confirm structures [11], to detect chemical modifications [2,10,18], and to identify sequence variations [14, 19 -21]. However, the deduction of sequence information is time-consuming, highly technical, lacking strict objectivity due to reliance on human interpretation, and can be performed only in laboratories with extensive experience in MSMS. Hence, automation of pro...