Using Protein Dimers to Maximize the Protein Hybridization Efficiency with Multisite DNA Origami Scaffolds.
DNA origami provides a versatile platform for conducting 'architecture-function' analysis to determine how the nanoscale organization of multiple copies of a protein component within a multi-protein machine affects its overall function. Such analysis requires that the copy number of protein molecules bound to the origami scaffold exactly matches the desired number, and that it is uniform over an entire scaffold population. This requirement is challenging to satisfy for origami scaffolds with many protein hybridization sites, because it requires the successful completion of multiple, independent hybridization reactions. Here, we show that a cleavable dimerization domain on the hybridizing protein can be used to multiplex hybridization reactions on an origami scaffold. This strategy yields nearly 100% hybridization efficiency on a 6-site scaffold even when using low protein concentration and short incubation time. It can also be developed further to enable reliable patterning of a large number of molecules on DNA origami for architecture-function analysis.
Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, Michigan, United States of America.
Vikash
V
Verma
Life Sciences Institute and Department of Biological Chemistry, University of Michigan Medical School, Michigan, United States of America.
Leena
L
Mallik
Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, Michigan, United States of America.
Rizal F
RF
Hariadi
Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, Michigan, United States of America.
Sivaraj
S
Sivaramakrishnan
Life Sciences Institute and Department of Biological Chemistry, University of Michigan Medical School, Michigan, United States of America.
Georgios
G
Skiniotis
Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, Michigan, United States of America.
Ajit P
AP
Joglekar
D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000235:genetics; D005978:Glutathione / Q000737:chemistry; D005990:Glycerol / Q000737:chemistry; D018386:Kinetochores / Q000737:chemistry; D007834:Lasers; D008854:Microscopy, Electron; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics; D009693:Nucleic Acid Hybridization / Q000379:methods*; D009841:Oligonucleotides / Q000737:chemistry* / Q000235:genetics; D055503:Protein Multimerization; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics; D015003:Yeasts / Q000235:genetics
D004271:DNA, Fungal; D004277:DNA, Single-Stranded; C112472:NDC80 protein, S cerevisiae; D009687:Nuclear Proteins; D009841:Oligonucleotides; D029701:Saccharomyces cerevisiae Proteins; D005978:Glutathione; D005990:Glycerol