Activity of Lambda-Exonuclease on Surface-Attached Oligonucleotide Detected by Acoustic Wave Device and Radiochemical Labeling

Abstract

Single-strand 25-mer oligonucleotides have been attached to the surface of a 9-MHz acoustic wave sensor incorporated into a flow-through configuration. This was achieved by an online interaction of the biotinylated probe with chemisorbed neutravidin. Duplex formation with complementary 25-mer was monitored by acoustic wave detection in real time. Regeneration of the probe-modified surface was achieved using online introduction of λ-exonuclease, an enzyme that digests a single strand of double-stranded DNA beginning with the 5′ end, cleaving single nucleotides as it progresses. Thus, with the 5′ end of the probe strand remaining inaccessible because of the biotin-neutravidin linkage, the target strand was successfully removed by enzymatic digestion. The results of the acoustic wave work are correlated with analogous experiments involving oligonucleotides labeled with ³²P.

Keywords:

• Acoustic wave device
• biosensors
• λ-exonuclease
• flow-through format
• nucleic acid ³²P labeling
• sensor regeneration

The authors are grateful to the Natural Sciences and Engineering Research Council of Canada for support of this work. Also, we thank Jon Ellis of the Institute of Biomaterials and Biomedical Engineering of the University of Toronto for very helpful discussions regarding the physics of acoustic wave propagation and assistance in the preparation of the manuscript.

Notes

Note. Additions of labeled F2 are given in bold type.

^a Labeled F2 is designated F2∗. Conditions are A: F2 (39 pmol), exo (90 min); B: F2 (735 pmol), exo (90 min); C: F2 (750 pmol), exo (15 min); D: F2 (1490 pmol), exo (15) min.