Chemically functionalized carbon films for single molecule imaging

Marc C. Llaguno, Hui Xu, Liang Shi, Nian Huang, Hong Zhang, Qinghua Liu, Qiu Xing Jiang

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

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 15. Å 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.

Original languageEnglish (US)
Pages (from-to)405-417
Number of pages13
JournalJournal of Structural Biology
Volume185
Issue number3
DOIs
StatePublished - Mar 2014

Fingerprint

Carbon
Gi-Go GTP-Binding Protein alpha Subunits
RNA
Ligands
Macromolecular Substances
RNA Interference
Oligonucleotides
Detergents
Catalytic Domain
Coloring Agents
Salts
Electrons
Temperature
Single Molecule Imaging
DNA
Enzymes

Keywords

  • Biological ligands
  • C3PO/RNA complex
  • Carbon films
  • ChemiC
  • Chemical functionalization
  • Single particle cryoEM

ASJC Scopus subject areas

  • Structural Biology

Cite this

Chemically functionalized carbon films for single molecule imaging. / Llaguno, Marc C.; Xu, Hui; Shi, Liang; Huang, Nian; Zhang, Hong; Liu, Qinghua; Jiang, Qiu Xing.

In: Journal of Structural Biology, Vol. 185, No. 3, 03.2014, p. 405-417.

Research output: Contribution to journalArticle

Llaguno, Marc C. ; Xu, Hui ; Shi, Liang ; Huang, Nian ; Zhang, Hong ; Liu, Qinghua ; Jiang, Qiu Xing. / Chemically functionalized carbon films for single molecule imaging. In: Journal of Structural Biology. 2014 ; Vol. 185, No. 3. pp. 405-417.
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