DNA stability in the gas versus solution phases: A systematic study of thirty-one duplexes with varying length, sequence, and charge level

Abstract

We report herein a systematic mass spectrometric study of a series of thirty-one non-selfcomplementary, matched, DNA duplexes ranging in size from 5-to 12-mers. The purpose of this work is threefold: (1) to establish the viability of using mass spectrometry as a tool for examining solution phase stabilities of DNA duplexes; (2) to systematically assess gas-phase stabilities of DNA duplexes; and (3) to compare gas and solution phase stabilities in an effort to understand how media affects DNA stability. These fundamental issues are of importance both on their own, and also for harnessing the potential of mass spectrometry for biological applications. We have found that ion abundances do not always track with solution phase stability; GC content must be taken into account. Two duplexes with the same T m yet with differing GC content can yield different ion abundances. That is, if two duplexes have the exact same melting temperature, yet one has a higher GC content, the duplex with the higher GC content yields a higher ion abundance. It thus appears that not only is a GC base pair stronger than an AT base pair, but the relative strengths of each differ in the gas phase versus in solution, such that the electrospray process can differentiate between them. We also characterize the gas-phase stabilities of the duplexes, using collision-induced dissociation (CID) as a method to assess stability. We focus on two aspects of this CID experiment. One, we examine what factors appear to control whether the duplexes dissociate into single strands or covalently fragment; we are able to utilize a charge state normalization we coin "charge level" to compare our results with others' and establish generalities regarding dissociation versus fragmentation patterns. Two, we examine those duplexes that primarily dissociate and use CID to assess the gas-phase stabilities. We find that correlation of gas-phase to solution-phase stabilities is more likely to occur when duplexes of varying GC content are examined. Duplexes with the same GC content tend to have stabilities that do not parallel those in solution. We discuss these results in light of the different roles that hydrogen bonding and base stacking play in solution versus the gas phase. Ultimately, we apply what we learn to lend insight into the biological problem of how the carcinogenic, damaged nucleobase O 6 -methylguanine causes mutations. (J Am Soc Mass Spectrom 2006, 17, 1383-1395

The Contribution of the Activation Entropy to the Gas-Phase Stability of Modified Nucleic Acid Duplexes

“…Previous studies showed that the formation of backbone fragments is promoted by a high GC content in the DNA duplex, and therefore, it is considered indicative of high gasphase stability [18]. Comparison of the four nonmodified duplexes in Table 1 reveals that this does not hold true for RNA duplexes.…”

“…Alternatively, the stability of a duplex can be evaluated with the E 50 value, which corresponds to the activation energy required to dissociate 50% of the duplex. In general, the E 50 value and the melting temperature T m measured in solution correlate for duplexes of similar GC content and identical size, which indicates that hydrogen bonding and base stacking are conserved in the gas phase [17,18]. Although similar relationships between solution and gas-phase stability were demonstrated for DNA and RNA duplexes, there is no comprehensive study that directly compares different types of nucleic acid duplexes.…”

“…High charge states promote strand separation [19] and favor asymmetric charge distribution within the duplex [20]. The formation of backbone fragments, on the other hand, is promoted by high GC content [18] and generally indicates high gas-phase stability of the duplex. Double resonance experiments show that as in single strands, neutral base loss precedes backbone cleavage in the duplex [21].…”

Non-covalent complexes between single-stranded oligodeoxynucleotides and poly(ethylene imine)

“…Relative abundance was calculated by dividing the complex intensity, I(complex 2+ ), by the sum I(complex 2+ ) + 1/2 [I(PEI + ) + I(ODN + )] [12,13]; E lab was calculated from the applied laboratory-frame collision energy (E lab ) using the equation E cm = E lab × (m Ar /(m Ar + m precursor )), where m Ar and m precursor designate the masses of Ar atoms and the precursor ion, respectively. The points were fitted into sigmoid curves, constructed using the Origin 8.1 graphing software [14], in order to deduce the corresponding E 50 values, which are the collision energies at which 50% of the PEI-ODN precursor ions were depleted due to CAD [12,13,15]. The E 50 value derived for each PEI-ODN complex is an average of three measurements.…”

Structures, fragmentation, and protonation of trideoxynucleotide CCC mono- and dianions

“…T he advent of electrospray ionization (ESI) [I, 2] has spawned remarkable advances in the structural characterization of biomolecules, including DNA, RNA, and oligodeoxyI ribonucleotides, as well as their metallated adducts and noncovalent complexes with proteins and drugs [3][4][5][6][7][8][9][10][11][12][13][14][15]. Both positive and negative gas-phase ions of oligodeoxynucleotides can now be formed at will, covering a range of charge states according to chosen electrospray conditions.…”