Hybridization and enzymatic extension of au nanoparticle-bound oligonucleotides.
We have investigated the impact of steric effects on the hybridization and enzymatic extension of oligonucleotides bound to 12-nm colloidal Au particles. In these experiments, a nanoparticle-bound 12-mer sequence is hybridized either to its solution phase 12-mer complement or to an 88-mer template sequence. The particle-bound oligonucleotide serves as a primer for enzymatic extension reactions, in which covalent incorporation of nucleotides to form the complement of the template is achieved by the action of DNA polymerase. Primers were attached via-C(6)H(12)SH, -C(12)H(24)SH, and -TTACAATC(6)H(12)SH linkers attached at the 5' end. Primer coverage on the nanoparticles was varied by dilution with (5')HSC(6)H(12)AAA AAA(3'). Hybridization efficiencies were determined as a function of linker length, primer coverage, complement length (12-mer vs 88-mer), and primer:complement concentration ratio. In all cases, hybridization for the 88-mer was less efficient than for the 12-mer. Low primer surface coverage, greater particle-primer separation, and higher primer:complement ratios led to optimal hybridization. Hybridization efficiencies as high as 98% and 75% were observed for the 12-mer and 88-mer, respectively. Enzymatic extension of particle-bound primers was observed under all conditions tested; however, the efficiency of the reaction was strongly affected by linker length and primer coverage. Extension of primers attached by the longest linker was as efficient as the solution-phase reaction.
Nicewarner Peña
Sheila R
SR
Department of Chemistry and Life Sciences Consortium, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Raina
Surabhi
S
Goodrich
Glenn P
GP
Fedoroff
Nina V
NV
Keating
Christine D
CD
D004247:DNA / Q000737:chemistry / Q000378:metabolism; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism; D006046:Gold / Q000737:chemistry*; D036103:Nanotechnology; D009693:Nucleic Acid Hybridization / Q000379:methods*; D009841:Oligonucleotides / Q000737:chemistry*; D010316:Particle Size
D017931:DNA Primers; D009841:Oligonucleotides; D006046:Gold; D004247:DNA; D004259:DNA-Directed DNA Polymerase