5 research outputs found
Cation binding to nucleic acids
No full textInternational audienceIntroductionDetecting cation and metal adducts to nucleic acids by mass spectrometry is easy. Too easy. Cations present in solution (intentionally or not) indeed stick very well to nucleic acid multiply charged ions. In the negative ion mode, metal cations cannot be removed by collisional activation. What is difficult is therefore not to detect cation binding, but to distinguish specific cation binding at peculiar coordination sides (which are often relevant on the structural biology point of view), from nonspecific cation âadductsâ at randomly distributed sites. Here we discuss strategies to distinguish specific from nonspecific binding, to quantify specifically bound cations. We will also discuss the origin of nonspecific adduct formation.MethodsDNA sequences forming single-stranded or G-quadruplex structures (which require specifically bound potassium ions to form), and RNA sequences forming single strands, hairpins, duplexes or kissing loop complexes (which require specifically bound magnesium ions to form) were purchased from Eurogentec or IDT. Magnesium or manganese acetate, KCl, ammonium acetate or trimethylammonium acetate were used to fix ionic strength and cation concentration. Samples were analyzed by an Agilent 6560 electrospray-IMS-Q-TOF, which allows to record drift tube ion mobility data from each m/z. The nonspecific adducts distributions were derived from control sequences, as a function of the charge state (including in supercharging conditions obtained with sulfolane). Then, the specific adducts distributions were quantified after subtraction of the nonspecific adducts contribution.Preliminary Data Here nonspecific adducts are defined as metal adducts formed upon binding to groups or structures that are not the specific structural motif under investigation. It does not mean that these adducts (or ion pairs) do not exist in solution. For example, for G-quadruplexes, the specific adducts are the potassium adducts bound to the specific inter-quartet locations, while the nonspecific adducts are those that would form in any single stranded sequence of the same length and base content. In the first part of the presentation, we will discuss the charge state dependence of the nonspecific adducts. If the distribution of adducts in the mass spectra depends on the charge state, we know that adducts formed during the electrospray process (and not just those formed in solution) do contribute to the overall distribution. In purely aqueous solvents, the number of nonspecific adducts decreases when z increases. But surprisingly, upon supercharging with sulfolane, more nonspecific adducts (of cations!) are observed on the extra highly (negatively!) charged ions. This counterintuitive observation actually gives insight on the mechanism of multiply charged ion production. In the second part of the presentation, we will discuss how to subtract the contribution of nonspecific adducts to deduce the fraction of metal ions specifically bound to particular structures such as the G-quadruplexes or RNA kissing loop motifs, and deduce the number of specific binding sites and the equilibrium binding constants, or metal binding rate constants. Finally, we will show how ion mobility spectrometry contributes discerning specific structures formed upon metal cation binding. These techniques were used to decipher the complex potassium-induced folding pathways of telomeric DNA G-quadruplex structures. Novel Aspect:We clarify here the origin of nonspecific adducts formation, and explain how to account for them and contribute suppressing them
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