Metal Cation Mediated-Capillary Electrophoresis of Nucleic Acids

Abstract

Nucleic acid electrophoresis separation heavily depends upon gel or nongel sieving matrix. Here we propose a metal ion mediated-capillary electrophoresis (MCE-CE) by utilizing the nonspecific interactions of Mg 2+ and Ca 2+ and demonstrate the size, conformation, or sequence based separation and characterization of versatile nucleic acid molecules in free solution. Mg 2+ and Ca 2+ can induce DNA separation at the concentrations as low as 100 and 50 µM, respectively. Noteworthy, the two naturally occurring polymorphisms of one base substitution that may change the secondary folding structure or base stacking can be discriminated by MCM-CE, showing its unique capability of resolving length-identical but conformation-different ssDNA. Benefiting from the achieved separation, we further demonstrate that the folding conformation of oligomers and its change caused by single base substitution can be promptly sensed by online coupled fluorescence polarization. We anticipate that this method will be applicable in length polymorphism analysis, singlestrand polymorphism analysis, hybridization analysis, microRNA analysis, and study of protein-nucleic acid interactions and the conformation-function relationship.Beyond the well-known coding of inheritable information by the primary structure of nucleic acids, recent landmark discoveries reveal that versatile nucleic acid molecules function as regulation of gene expression (RNA interference, microRNA, riboswitch, DNA methylation), recognition of specific substrates (aptamer), and/or catalysis (DNAzyme, ribozyme, aptazyme), which may be underlined by their secondary or tertiary structures. 1 Although sieving matrix-dependent capillary electrophoresis can provide high throughput and accurate sequencing for the primary structure and methylation of DNA now, 2-5 there is a lack of an efficient separation means to identify and characterize complex secondary and tertiary structures of nucleic acids in free solution. In traditional electrophoresis (as gel or nongel sieving electrophoresis), nucleic acid molecules are often required to interact with the polymer based sieving matrix, 6,7 which may interfere with their native conformations. Free solution capillary electrophoresis is ideal for conformational separation and analysis because of the preservation of native conformations of nucleic acids. Although nucleic acids are negatively charged, the separation of oligonucleotides in free solution was only observed up to 10 bases because of the well-known small difference in electrophoretic mobility for nucleic acids as the base number increases. [8][9][10][11] The sizeseparation may be achieved through the modification of DNA by a neutral drag-tag polymer 12 or DNA binding proteins. 13,14 With the use of a buffer system containing zinc and sodium dodecyl sulfate (SDS) micelle, it is possible to separate 14 out of 18 oligonucleotides of 8 bases. 8 The specific interactions of metal ions and oligonucleotides (e.g., K + and G-quadruplex and Ni 2+ and guanosine) were also used to separate...

Highly sensitive determination of recombinant human erythropoietin-α in aptamer-based affinity probe capillary electrophoresis with laser-induced fluorescence detection

“…Previous research reported that some cations played essential roles on specific interaction between protein and DNAs [13,17,18]. Meanwhile, some ions presented in the running buffer could also affect the electroosmotic flow (EOF) and consequently improve the resolution of proteins, DNAs and other biomacromolecules [24][25][26]. The effect of kinds of anions or cations in the sample or running buffer on the separation efficiency was investigated.…”

Affinity capillary electrophoresis with laser induced fluorescence detection for thrombin analysis using nuclease-resistant RNA aptamers

“…The addition of 1 mM MgCl 2 in TG separation buffer caused the complex peak and free aptamer peak disappeared in the tested time range due to the change of EOF caused by MgCl 2 , which significantly slow down the separation [33]. With increase of NaCl in TG separation buffer, the complex peak became small (Fig.…”

“…A previously reported laboratory-built capillary electrophoresis laser induced fluorescence system was used for separation and detection [18,24,33]. Briefly, uncoated fused silica capillaries were used for separation (40 cm in length, 365 m o.d., 25 m.…”

Capillary electrophoresis applied to DNA: determining and harnessing sequence and structure to advance bioanalyses (2009–2014)

“…The presence of guanosine 5'-monophosphate, adenosine 5'-monophosphate, uridine 5'-monophosphate, deoxyguanosine monophosphate, or phosphate supported sequence-based selectivity of DNA fragments which was suggested as an alternative to stability and conformation-based analyses [ 155 ]. Metal cation mediated-capillary electrophoresis, which is sensitive to conformational change [ 156 ], generates separation-based aptamer assays of 5 μM cocaine detection through conformational change associated with displacement of the aptamer target [ 157 ]. DNA aptamers can also be used as labels for indirect molecular detection.…”