Massively Parallel Interrogation of Aptamer Sequence, Structure and Function

Journal Article · Fri Feb 08 04:00:00 EST 2008 · PLoS ONE, vol. 3, no. 7, July 16, 2008, e2720

DOI:https://doi.org/10.1371/journal.pone.0002720· OSTI ID:945600

Fischer, N O ^[1]; Tok, J B; Tarasow, T M

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1. DOE/OSTI

Optimization of high affinity reagents is a significant bottleneck in medicine and the life sciences. The ability to synthetically create thousands of permutations of a lead high-affinity reagent and survey the properties of individual permutations in parallel could potentially relieve this bottleneck. Aptamers are single stranded oligonucleotides affinity reagents isolated by in vitro selection processes and as a class have been shown to bind a wide variety of target molecules. Methodology/Principal Findings. High density DNA microarray technology was used to synthesize, in situ, arrays of approximately 3,900 aptamer sequence permutations in triplicate. These sequences were interrogated on-chip for their ability to bind the fluorescently-labeled cognate target, immunoglobulin E, resulting in the parallel execution of thousands of experiments. Fluorescence intensity at each array feature was well resolved and shown to be a function of the sequence present. The data demonstrated high intra- and interchip correlation between the same features as well as among the sequence triplicates within a single array. Consistent with aptamer mediated IgE binding, fluorescence intensity correlated strongly with specific aptamer sequences and the concentration of IgE applied to the array. The massively parallel sequence-function analyses provided by this approach confirmed the importance of a consensus sequence found in all 21 of the original IgE aptamer sequences and support a common stem:loop structure as being the secondary structure underlying IgE binding. The microarray application, data and results presented illustrate an efficient, high information content approach to optimizing aptamer function. It also provides a foundation from which to better understand and manipulate this important class of high affinity biomolecules.

• Research Organization: Lawrence Livermore National Laboratory (LLNL), Livermore, CA
• Sponsoring Organization: USDOE
• DOE Contract Number: W-7405-ENG-48
• OSTI ID: 945600
• Report Number(s): LLNL-JRNL-401442
• Journal Information: PLoS ONE, vol. 3, no. 7, July 16, 2008, e2720, Journal Name: PLoS ONE, vol. 3, no. 7, July 16, 2008, e2720 Journal Issue: 7 Vol. 3
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
AFFINITY
DNA
FLUORESCENCE
IMMUNOGLOBULINS
IN VITRO
MEDICINE
MICROARRAY TECHNOLOGY
OLIGONUCLEOTIDES
OPTIMIZATION
TARGETS