Signaling Aptamers Created Using Fluorescent Nucleotide Analogues

Abstract

A new approach to creating fluorescent signaling aptamers using fluorescent nucleotide analogues is presented. The fluorescence quantum yield of nucleotide analogues such as 2-aminopurine strongly depends on base stacking interactions when incorporated into double or single stranded DNA. This property is used to generate a binding-specific fluorescence signal. Aptamers for human alpha-thrombin, immunoglobulin E, and platelet-derived growth factor B were modified with fluorescent nucleotide analogues in positions that undergo conformational changes. The resulting signaling aptamers show a specific, binding-induced increase in the fluorescence signal of up to 30-fold. Conformation-changing positions in these aptamers were identified by screening a set of modified aptamer sequences that each included a fluorescent nucleotide analogue at a different position. The positions for these modifications were estimated by modeling the aptamer secondary structure. It is likely that this approach to producing fluorescent signaling aptamers is of general use for protein-binding aptamers because of their "induced fit" binding mechanism.

Fluorescent sensing ochratoxin A with single fluorophore-labeled aptamer

“…Usually, fluorescent dyes can be readily introduced on the ends of the aptamers through chemical synthesis to fabricate the aptamer switch [5][6][7][9][10][11][12][13][14]. It is also feasible to conjugate fluorophores on the internal bases or to use the fluorescent analogue of the bases in oligonucleotides for sensing [5][6][7][15][16][17][18].…”

A fluorescein labeled aptamer switch for thrombin with fluorescence decrease response

“…Compared with a few reported aptamer-based uorescence assays for thrombin, this assay has more simple steps and a lower limit (Table S2 †). 10,11,13,[15][16][17][18] Specicity test…”

Rapid fluorescence detection of immunoglobulin E using an aptamer switch based on a binding-induced pyrene excimer

“…21 The discovery of aptamers for IgE allows the development of aptamer-based assays for IgE in different formats. 12,15,[17][18][19][20][22][23][24][25][26][27][28] To date, several aptamer-based optical detection methods (e.g. luminescence and uorescence) have been developed for IgE detection, [22][23][24][25][26][27][28] such as a label-free aptasensor based on a molecular light switch, 22 uorescence anisotropy assay relying on IgE-binding induced increase of molecular volume and uorescence anisotropy, 23 competitive molecular beacon assay, 26 etc.…”