A Broadly Applicable Assay for Rapidly and Accurately Quantifying DNA Surface Coverage on Diverse Particles.
DNA-modified particles are used extensively for applications in sensing, material science, and molecular biology. The performance of such DNA-modified particles is greatly dependent on the degree of surface coverage, but existing methods for quantitation can only be employed for certain particle compositions and/or conjugation chemistries. We have developed a simple and broadly applicable exonuclease III (Exo III) digestion assay based on the cleavage of phosphodiester bonds-a universal feature of DNA-modified particles-to accurately quantify DNA probe surface coverage on diverse, commonly used particles of different compositions, conjugation chemistries, and sizes. Our assay utilizes particle-conjugated, fluorophore-labeled probes that incorporate two abasic sites; these probes are hybridized to a complementary DNA (cDNA) strand, and quantitation is achieved via cleavage and digestion of surface-bound probe DNA via Exo III's apurinic endonucleolytic and exonucleolytic activities. The presence of the two abasic sites in the probe greatly speeds up the enzymatic reaction without altering the packing density of the probes on the particles. Probe digestion releases a signal-generating fluorophore and liberates the intact cDNA strand to start a new cycle of hybridization and digestion, until all fluorophore tags have been released. Since the molar ratio of fluorophore to immobilized DNA is 1:1, DNA surface coverage can be determined accurately based on the complete release of fluorophores. Our method delivers accurate, rapid, and reproducible quantitation of thiolated DNA on the surface of gold nanoparticles, and also performs equally well with other conjugation chemistries, substrates, and particle sizes, and thus offers a broadly useful assay for quantitation of DNA surface coverage.
Department of Chemistry and Biochemistry, Florida International University , 11200 SW Eighth Street, Miami, Florida 33199, United States.
Haixiang
H
Yu
Department of Chemistry and Biochemistry, Florida International University , 11200 SW Eighth Street, Miami, Florida 33199, United States.
Xiaowen
X
Xu
Department of Chemistry and Biochemistry, Florida International University , 11200 SW Eighth Street, Miami, Florida 33199, United States.
Pingping
P
Liang
Department of Chemistry, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
Kang Yong
KY
Loh
Department of Chemistry and Biochemistry, Florida International University , 11200 SW Eighth Street, Miami, Florida 33199, United States.
Bhargav
B
Guntupalli
Department of Chemistry and Biochemistry, Florida International University , 11200 SW Eighth Street, Miami, Florida 33199, United States.
Daniel
D
Roncancio
Department of Chemistry and Biochemistry, Florida International University , 11200 SW Eighth Street, Miami, Florida 33199, United States.
Yi
0000-0001-7278-9811
Y
Xiao
D001483:Base Sequence; D015342:DNA Probes / Q000032:analysis* / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D006046:Gold / Q000737:chemistry*; D061925:Immobilized Nucleic Acids / Q000032:analysis* / Q000378:metabolism; D053768:Metal Nanoparticles / Q000737:chemistry*; D009693:Nucleic Acid Hybridization / Q000379:methods; D055672:Static Electricity; D013499:Surface Properties
D015342:DNA Probes; D005456:Fluorescent Dyes; D061925:Immobilized Nucleic Acids; D006046:Gold; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III