Capillary Electrophoresis–Systematic Evolution of Ligands by Exponential Enrichment Selection of Base- and Sugar-Modified DNA Aptamers: Target Binding Dominated by 2′-O,4′-C-Methylene-Bridged/Locked Nucleic Acid Primer

Abstract

Chemically modified DNA aptamers specific to human α-thrombin were obtained from oligodeoxyribonucleotide (ODN) libraries by using a capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX) method. These libraries contained 2'-O,4'-C-methylene-bridged/linked bicyclic ribonucleotides (B/L nucleotides) in the primer region and/or C5-modified thymidine bearing N(6)-ethyladenine (t) in the nonprimer region. Modified DNA aptamers showed high binding affinities to the target, with dissociation constants (Kd) values in the range of subnanomolar to several ten nanomolar levels. The introduction of base modification significantly suppressed the frequency of G-quadruplex motifs, which are often seen in thrombin-binding DNA aptamers. The resulting alternatives contained the 10-mer consensus sequence t5Gt2G2, which is frequently found in modified DNA aptamers with subnanomolar protein binding affinities. Furthermore, some base- and sugar-modified DNA aptamers with the 12-mer consensus sequence t2G2tC(A/G)A2G2t displayed binding activities that were dependent on the presence of B/L nucleotides in the primer region. Such aptamers were interestingly not recovered from a natural DNA library or from DNA libraries modified with either B/L nucleotides or t's. This emerging characteristic binding property will enable the creation of a direct selection methodology for DNA-based molecular switches that are triggered by chemical conversion of B/L nucleotides introduced to constant sequence regions in ODN libraries.

Using G-Quadruplex/Hemin To “Switch-On” the Cathodic Photocurrent of p-Type PbS Quantum Dots: Toward a Versatile Platform for Photoelectrochemical Aptasensing

“…[56][57][58] Because the band resulted from the recognition of TB by TBA was difficult to be identified by electrophoresis, 59 we employed UV-vis absorption and CD spectra to measure the structure and the binding of hemin and TB to TBA. 60,61,62 As shown in Figure S8A, when mixing TBA and hemin, the typical absorption of hemin became stronger with the absorption peak red shifted from 395 to 404 nm (curve a and b), indicating the formation of a G-quadruplex/hemin structure. 46 The presence of TB caused a further hyperchromicity of the hemin (curve c), which demonstrated that TB promoted binding of hemin in G-quadruplex.…”

“…61 In the presence of TB, the peak intensity of Gquadruplex/hemin increased at 292 nm (curve c). This implied that the pattern of the G-quadruplex structure did not change, but the antiparallel structure became more compact by TB.…”

New Technologies Provide Quantum Changes in the Scale, Speed, and Success of SELEX Methods and Aptamer Characterization

“…In addition to previously reported selection of ssDNA and RNA aptamers, successful selection of base- and sugar modified DNA aptamers via CE-SELEX has been reported for human α-thrombin. 43 Small size of the starting library in CE-SELEX (~10 12 sequences), a major limitation, has been improved significantly by using a micro free flow electrophoresis (μFFE) device (300-fold, 1.8 10 14 sequences). 44 Isolation of nanomolar affinity aptamers after a single round ( i.e.…”

The expanding world of DNA and RNA

“…Therminator DNA polymerase was used to extend a DNA hairpin primer/template with the modified nucleotides, permitting amplification of the DNA primer-template after selection and resynthesis of the selected TNA aptamer. Kuwahara and co-workers used a mixture of mutant and/or wild type KOD DNA polymerases to “transcribe” and “reverse transcribe” mixed LNA/DNA oligonucleotides during selection of an anti-thrombin aptamer [15,16]. Holliger and co-workers have dramatically expanded the potential of these analogs by evolving a series of TgoT DNA polymerase mutants to better “transcribe” and “reverse transcribe” these modified nucleotides, and they have used them to select HNA aptamers targeting HIV trans-activating response RNA and hen egg lysozyme [17•], a FANA aptamer targeting HIV-1 RT [18], and catalytic nucleic acids containing ANA, FANA, HNA, and CeNA moieties [19].…”