Selection of structure-induced aptamer targeting small molecule based on capillary sieving electrophoresis.

In this study, a structure-induced aptamer targeting small molecules was selected using capillary sieving electrophoresis (CSE). CSE was conducted using a capillary filled with a background solution containing hydroxypropyl cellulose as a sieving matrix to separate the aptamer candidates by changing their structures via complexation. Before aptamer selection, the original random-sequence DNA library was used to create structure-not-preorganized DNA sub-library containing straight-chain-like structures using CSE. Next, a structure-induced aptamer targeting L-tyrosinamide was selected from the prepared sub-library. Six aptamer candidates were selected, one of which showed a binding ability comparable to that of the reported L-tyrosinamide aptamer and selectivity toward the analogs. These results indicated that the proposed method can be applied to select structure-induced aptamers that target small molecules.

Assessment of the structural change of DNA by binding with a small molecule based on capillary sieving electrophoresis.

In this study, capillary sieving electrophoresis (CSE) using polymer solutions was used to evaluate the structural changes in nucleic acids upon complexation with small molecules. As the model target and nucleic acids, L-tyrosinamide (Tyr-Am) and its aptamer, which is a type of DNA specifically binding to Tyr-Am, were selected. CSE was conducted using a capillary filled with background solution (BGS) containing hydroxypropyl cellulose (HPC) as a sieving matrix. When Tyr-Am or tyrosine was added to the BGS in CSE, the ratio of mobility differences of the Tyr-Am-aptamer complex increased compared to that of the free aptamer without the addition of Tyr-Am. In contrast, when other amino acids or their analogs were added, results showed no apparent change or decreases in electrophoretic mobility. These results indicate that the proposed method can be applied to assess structural changes in nucleic acids that target small molecules.

Fractionation of Single-stranded DNAs with/without Stable Preorganized Structures Using Capillary Sieving Electrophoresis for Aptamer Selection.

Aptamers, single-stranded DNAs/RNAs with a strong and specific interaction towards a target molecule, have wide applications in the fields of medicine and biosensors. In conventional aptamer selection methods, it is difficult to obtain "preorganized" and/or "induced-fit" type of aptamers selectively. In this study, separation and fractionation of single-stranded DNAs with/without stable preorganized structures were carried out using capillary sieving electrophoresis. The fractionated DNAs showed different mobilities and thermodynamic stabilities of their secondary structures; this outcome is deemed to be necessary for the synthesis of novel aptasensors with a desirable sensing mechanism.