Open AccessImmobilization of Lambda Exonuclease onto Polymer Micropillar Arrays for the Solid-Phase Digestion of dsDNAs
Author(s) -
Nyoté J. Oliver-Calixte,
Franklin I. Uba,
Katherine E. Battle,
Kumuditha M. Weerakoon-Ratnayake,
Steven A. Soper
Publication year - 2014
Publication title -
analytical chemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.117
H-Index - 332
eISSN - 1520-6882
pISSN - 0003-2700
DOI - 10.1021/ac5002965
Subject(s) - chemistry , exonuclease , klenow fragment , combinatorial chemistry , exonuclease iii , digestion (alchemy) , dna , polymer , phase (matter) , enzyme , chemical engineering , chromatography , organic chemistry , biochemistry , polymerase , escherichia coli , gene , engineering
The process of immobilizing enzymes onto solid supports for bioreactions has some compelling advantages compared to their solution-based counterpart including the facile separation of enzyme from products, elimination of enzyme autodigestion, and increased enzyme stability and activity. We report the immobilization of λ-exonuclease onto poly(methylmethacrylate) (PMMA) micropillars populated within a microfluidic device for the on-chip digestion of double-stranded DNA. Enzyme immobilization was successfully accomplished using 3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) coupling to carboxylic acid functionalized PMMA micropillars. Our results suggest that the efficiency for the catalysis of dsDNA digestion using λ-exonuclease, including its processivity and reaction rate, were higher when the enzyme was attached to a solid support compared to the free solution digestion. We obtained a clipping rate of 1.0 × 10(3) nucleotides s(-1) for the digestion of λ-DNA (48.5 kbp) by λ-exonuclease. The kinetic behavior of the solid-phase reactor could be described by a fractal Michaelis-Menten model with a catalytic efficiency nearly 17% better than the homogeneous solution-phase reaction. The results from this work will have important ramifications in new single-molecule DNA sequencing strategies that employ free mononucleotide identification.