Chemically functionalized carbon films for single molecule imaging.
Many biological complexes are naturally low in abundance and pose a significant challenge to their structural and functional studies. Here we describe a new method that utilizes strong oxidation and chemical linkage to introduce a high density of bioactive ligands onto nanometer-thick carbon films and enable selective enrichment of individual macromolecular complexes at subnanogram levels. The introduced ligands are physically separated. Ni-NTA, Protein G and DNA/RNA oligonucleotides were covalently linked to the carbon surface. They embody negligible mass and their stability makes the functionalized films able to survive long-term storage and tolerate variations in pH, temperature, salts, detergents, and solvents. We demonstrated the application of the new method to the electron microscopic imaging of the substrate-bound C3PO, an RNA-processing enzyme important for the RNA interference pathway. On the ssRNA-linked carbon surface, the formation of C3PO oligomers at subnanomolar concentrations likely mimics their assembly onto ssRNA substrates presented by their native partners. Interestingly, the 3D reconstructions by negative stain EM reveal a side port in the C3PO/ssRNA complex, and the 15Å cryoEM map showed extra density right above the side port, which probably represents the ssRNA. These results suggest a new way for ssRNAs to interact with the active sites of the complex. Together our data demonstrate that the surface-engineered carbon films are suitable for selectively enriching low-abundance biological complexes at nanomolar level and for developing novel applications on a large number of surface-presented molecules.
Department of Cell Biology, UT Southwestern Medical Center, 6000 Harry Hines Blvd, Dallas, TX 75390, USA.
Marc C
MC
Llaguno
Department of Cell Biology, UT Southwestern Medical Center, 6000 Harry Hines Blvd, Dallas, TX 75390, USA.
Hui
H
Xu
Department of Cell Biology, UT Southwestern Medical Center, 6000 Harry Hines Blvd, Dallas, TX 75390, USA.
Liang
L
Shi
Department of Biochemistry, UT Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390, USA; Department of Biophysics, UT Southwestern Medical Center, 6000 Harry Hines Blvd, Dallas, TX 75390, USA.
Nian
N
Huang
Department of Biochemistry, UT Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390, USA; Department of Biophysics, UT Southwestern Medical Center, 6000 Harry Hines Blvd, Dallas, TX 75390, USA.
Hong
H
Zhang
Department of Biochemistry, UT Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390, USA.
Qinghua
Q
Liu
Department of Cell Biology, UT Southwestern Medical Center, 6000 Harry Hines Blvd, Dallas, TX 75390, USA. Electronic address: qiu-xing.jiang@utsouthwestern.edu.
Qiu-Xing
QX
Jiang
D002244:Carbon / Q000737:chemistry*; D020285:Cryoelectron Microscopy; D004247:DNA / Q000737:chemistry; D006801:Humans; D036103:Nanotechnology; D012313:RNA / Q000737:chemistry*
D012313:RNA; D002244:Carbon; D004247:DNA
Biological ligands; C3PO/RNA complex; Carbon films; ChemiC; Chemical functionalization; Single particle cryoEM