A study of noncovalent complexes involving single-stranded DNA and polybasic compounds using nanospray mass spectrometry

Peran Terrier 0 1 Jeanine Tortajada 0 1 William Buchmann 0 1 0 Published online November 7, 2006 Address reprint requests to Dr. William Buchmann, Laboratoire Analyse et Modlisation pour la Biologie et l'Environnement, Universit d'Evry-Val d'Essonne , CNRS UMR 8587, Bt. Maupertuis, Bd. F. Mitterrand, 91025 Evry cdex, France 1 Laboratoire Analyse et Modlisation pour la Biologie et l'Environnement, Universit d'Evry-Val d'Essonne , CNRS UMR 8587, Evry, France Noncovalent complexes involving a single-stranded DNA oligonucleotide and a polybasic compound (spermine, penta-L-lysine, penta-L-arginine, or polydisperse poly-L-lysine) were detected by nanospray-MS. Several control experiments tended to show that these complexes preexisted in solution and that the interactions were initially ionic ones between oligonucleotide phosphates and protonated basic sites of the polybasic compound. Collision-induced dissociation (CID) experiments carried out with these complexes allowed us to identify some differences in the nature of the interactions between the solution and the gas phase, arising from possible proton transfers. Different dissociation pathways were observed according to the nature of the polybasic compound and to the initial charge state of the complex. The complex involving spermine dissociated by cleavage of noncovalent bonds leading to the separation of the two components, whereas the one involving penta-L-arginine underwent fragmentations of covalent bonds. Both behaviors were independent of the initial charge state of the complex. On the other hand, the dissociation pathway of the complex involving penta-L-lysine has been shown to be clearly charge state dependent. Noncovalent dissociation (separation of the two components) driven by coulomb repulsion occurred for the higher charged complexes, whereas fragmentation of covalent bonds was the main pathway of the lower charged complexes. In the latter case, differences in CID behavior were observed for different lengths of poly-L-lysine. (J Am Soc Mass Spectrom 2007, 18, 346 -358) 2007 American Society for Mass Spectrometry - Icharges and can interact with cationic molecules n living organisms, DNA phosphates bear negative through ionic bonds. Such bonds involving protonated basic amino acid residues [Lysine (Lys), Arginine (Arg), and Histidine (His)] play an important role in DNAprotein interactions. An example is the packaging of DNA in cell nuclei by histones, which are proteins including a high proportion of basic residues [1]. Moreover, some natural polyamines such as putrescine (NH2OC2H8ONH2), spermidine (NH2O C3H6ONHOC4H8ONH2), and spermine (NH2O C3H6ONHOC4H8ONHOC3H6ONH2) are present in cells as multi-protonated molecules and can interact with DNA [2]. Furthermore, complexes involving polybasic compounds and DNA have attracted new interest as a way to transport a therapeutic gene or an oligonucleotide in a cell. Poly-L-Lysine, polyamines, and their derivatives are extensively studied vectors [3, 4]. The development of electrospray ionization (ESI) [5] and matrix-assisted laser desorption/ionization (MALDI) [6, 7] allowed the emergence and rapid growth of the mass spectrometry of noncovalent complexes [8 11]. Complexes involving DNA are considerably less studied than those involving proteins and studies of the attachment between DNA and polyamines or polybasic peptides are particularly scarce. The only observations of natural polyamine oligonucleotide complexes by mass spectrometry have been reported in the case where spermine was used as matrix additives for oligonucleotide analysis by MALDITOF [1215]. In this case, protonated sites of the polyamine were expected to bond DNA phosphate in solution to displace alkaline cations [1214] and stabilize duplexes [15]. Ideally, polyamine protons were transferred to DNA phosphates during sample crystallization or desorption/ ionization process and the oligonucleotide free of alkaline cation and polyamine was observed. However, under some matrix conditions, unwanted polyamine oligonucleotide complexes were detected. Furthermore, polybasic peptide oligonucleotide complexes have also been detected by MALDI-TOF [1621].ObservationofsuchcomplexeswasfirstreportedbyJuhaszandBiemann[16].Inthatwork,highly acidic compounds (such as DNA) were detected as noncovalent complexes with a peptide or a small protein rich in Arginine, Lysine, and/or Histidine. Subsequently,Vertesetal.[17]studiedinteractionsbetween single-stranded oligonucleotide and basic dipeptides or smallproteins.Woodsetal.[18]formedcomplexes between basic peptides with various sequences and single/double-stranded oligonucleotides. By varying pH and comparing theoretical protonation state of free oligonucleotide nucleobases and phosphates with the presence or absence of complexes, interactions have been proven to occur between oligonucleotide phosphatesandpeptidebasicresiduesandtobeionic[17, 18].Anotherevidenceofthenatureoftheseinteractions was the observation of a competition between basic peptides and alkaline cations to bind the oligonucleotide phosphates: peaks of oligonucleotidepeptide complexes were sharper than those of free oligonucleotide, indicating a displacement of alkaline cations by basic peptides [17]. Conversely, when increasing ionic strength (by increasing ammonium salt concentration), ammonium cations displace basic peptides, resulting in the disappearance of the complex in the mass spectrum [18].Insomecases,variousstoichiometrieshavebeen observed, suggesting the absence of a specific binding site. Byusingdifferentoligonucleotides,Woodsetal.[18] confirmed that, at neutral pH, oligonucleotide base sequence does not seem to have any influence on the formation of the complex, whereas the composition of the peptide is important. First, the efficiency of complex formation depends obviously on the presence and numberofbasicresiduesinthepeptide[17].Interestingly, Arginine seems to have a greater role in the complex formationthanLysineandHistidine[17].Thiscanbe explained by a greater stability of its bond with the phosphate group in solution or in the gas phase resulting from its structure. Furthermore, the proportion and the arrangement of the basic amino acids within the peptide have been found to be important. Indeed, Woods et al. [18] observed that at least five basic residues in close proximity to each other, but separated by aliphatic residues, seemed to favor the formation of a complex with an oligonucleotide. This suggests that the geometry of the complex can play a role and/or that forces other than ionic ones are involved. Vasseur et al. [19]usedMALDI-TOFtoobservecomplexesbetween single-stranded oligonucleotide and guanidine derivatives (guanidine being the basic site of the arginine residues). They showed that the presence of an aromatic ring permitted a better interaction, pointing out the importance of complementary hydrophobic forces and -stacking. Such a peptide-sequence dependency has bee (...truncated)