title: Optimized light-directed synthesis of aptamer microarrays.
abstract: Aptamer microarrays are a promising high-throughput method for ultrasensitive detection of multiple analytes, but although much is known about the optimal synthesis of oligonucleotide microarrays used in hybridization-based genomics applications, the bioaffinity interactions between aptamers and their targets is qualitatively different and requires significant changes to synthesis parameters. Focusing on streptavidin-binding DNA aptamers, we employed light-directed in situ synthesis of microarrays to analyze the effects of sequence fidelity, linker length, surface probe density, and substrate functionalization on detection sensitivity. Direct comparison with oligonucleotide hybridization experiments indicates that aptamer microarrays are significantly more sensitive to sequence fidelity and substrate functionalization and have different optimal linker length and surface probe density requirements. Whereas microarray hybridization probes generate maximum signal with multiple deletions, aptamer sequences with the same deletion rate result in a 3-fold binding signal reduction compared with the same sequences synthesized for maximized sequence fidelity. The highest hybridization signal was obtained with dT 5mer linkers, and the highest aptamer signal was obtained with dT 11mers, with shorter aptamer linkers significantly reducing the binding signal. The probe hybridization signal was found to be more sensitive to molecular crowding, whereas the aptamer probe signal does not appear to be constrained within the density of functional surface groups commonly used to synthesize microarrays.
authors.affiliation: FlexGen, J.H. Oortweg 21, 2333CH Leiden, The Netherlands.
authors.forename: Nicole L W
authors.initials: NL
authors.lastname: Franssen-van Hal
authors.forename: Pepijn
authors.initials: P
authors.lastname: van der Putte
authors.forename: Klaus
authors.initials: K
authors.lastname: Hellmuth
authors.forename: Stefan
authors.initials: S
authors.lastname: Matysiak
authors.forename: Nicole
authors.initials: N
authors.lastname: Kretschy
authors.forename: Mark M
authors.initials: MM
authors.lastname: Somoza
mesh_terms: D052157:Aptamers, Nucleotide / Q000096:biosynthesis* / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D010775:Photic Stimulation / Q000379:methods*; D011485:Protein Binding / Q000502:physiology
chemical_list: D052157:Aptamers, Nucleotide