Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nanostructures.
Optical nanostructures have enabled the creation of subdiffraction detection volumes for single-molecule fluorescence microscopy. Their applicability is extended by the ability to place molecules in the confined observation volume without interfering with their biological function. Here, we demonstrate that processive DNA synthesis thousands of bases in length was carried out by individual DNA polymerase molecules immobilized in the observation volumes of zero-mode waveguides (ZMWs) in high-density arrays. Selective immobilization of polymerase to the fused silica floor of the ZMW was achieved by passivation of the metal cladding surface using polyphosphonate chemistry, producing enzyme density contrasts of glass over aluminum in excess of 400:1. Yields of single-molecule occupancies of approximately 30% were obtained for a range of ZMW diameters (70-100 nm). Results presented here support the application of immobilized single DNA polymerases in ZMW arrays for long-read-length DNA sequencing.
Korlach
Jonas
J
Pacific Biosciences, 1505 Adams Drive, Menlo Park, CA 94025, USA.
Marks
Patrick J
PJ
Cicero
Ronald L
RL
Gray
Jeremy J
JJ
Murphy
Devon L
DL
Roitman
Daniel B
DB
Pham
Thang T
TT
Otto
Geoff A
GA
Foquet
Mathieu
M
Turner
Stephen W
SW
D000535:Aluminum; D004270:DNA, Circular / Q000737:chemistry; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000302:isolation & purification; D004800:Enzymes, Immobilized / Q000737:chemistry*; D005898:Glass; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods; D049329:Nanostructures / Q000737:chemistry*; D055095:Optics and Photonics; D063065:Organophosphonates / Q000737:chemistry; D011145:Polyvinyls / Q000737:chemistry; D040081:Protein Array Analysis / Q000295:instrumentation* / Q000379:methods*; D013499:Surface Properties; D013698:Templates, Genetic
D004270:DNA, Circular; D004800:Enzymes, Immobilized; D063065:Organophosphonates; D011145:Polyvinyls; D000535:Aluminum; D012321:DNA-Directed RNA Polymerases