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BIO-MOLECUALR ADVANCES IN NANOTECHNOLOGY BY Viswaprakash Nanduri Instructor: Yonhua Tzeng

BIO-MOLECUALR ADVANCES IN NANOTECHNOLOGY BY Viswaprakash Nanduri Instructor: Yonhua Tzeng ELECT 7970 . OUTLINE: DEFINITION OF PHAGE BASIC TERMINOLOGY AND METHODS RELATED TO PHAGE 3. Phage display to align the nanoparticles 4. Self-Assembling DNA Nanostructures

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BIO-MOLECUALR ADVANCES IN NANOTECHNOLOGY BY Viswaprakash Nanduri Instructor: Yonhua Tzeng

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  1. BIO-MOLECUALR ADVANCES IN NANOTECHNOLOGY BY Viswaprakash Nanduri Instructor: Yonhua Tzeng ELECT 7970

  2. OUTLINE: • DEFINITION OF PHAGE • BASIC TERMINOLOGY AND METHODS RELATED TO PHAGE • 3. Phage display to align the nanoparticles • 4. Self-Assembling DNA Nanostructures • CHAPERONINS FOR THE FABRICATION OF QUANTUM DOTS

  3. What is phage? Filamentous phage M13, f1 and fd are thread-shaped bacterial viruses. Their outer coat is composed of thousands of 50-residue a-helical subunits of the major coat proteins, which overlap one another to form a tube encasing the viral DNA Courtesy: Dr.Valery Petrenko,Dept. of Pathobiology, Auburn University

  4. Reference: http://www.niddk.nih.gov/fund/other/genoproteo/wang.pdf

  5. Reference: http://www.niddk.nih.gov/fund/other/genoproteo/wang.pdf

  6. What are major coat and minor coat proteins? Foreign peptide/protein PIX PVII A phage-display library is a mixture of filamentousphage with foreign coding sequences spliced into pVIII, pIII or pVI. The peptide specified by the foreign DNA is displayed on the surface of the virion, fused to the coat protein. Reference: Adapted from “Phage Display-A laboratory manualby Carlos F. Barbas Single stranded genome PVIII PVI PIII

  7. pIII Phage display to align the nanoparticles A liquid crystal system was used for the fabrication of a highly ordered composite material from genetically engineered M13 ( on pIII coat protein) bacteriophage and zinc sulfide (ZnS) nanocrystals Semiconductor surface Phage library with 109 random peptide inserts 3 MAY 2002 VOL 296 SCIENCE www.sciencemag.org P892-895

  8. Phage display to align the nanoparticles Bioselection Semiconductor surface 3 MAY 2002 VOL 296 SCIENCE www.sciencemag.org P892-895

  9. Phage display to align the nanoparticles The bacteriophage,which formed the basis of the self-ordering system, were selected tohave a specific recognition moiety for ZnS crystal surfaces pH elution Semiconductor surface 3 MAY 2002 VOL 296 SCIENCE www.sciencemag.org P892-895

  10. Phage display to align the nanoparticles Semiconductor surface Enrichment by bacteria X 1,1000,000 for 4.5 hours 3 MAY 2002 VOL 296 SCIENCE www.sciencemag.org P892-895

  11. Phage display to align the nanoparticles Semiconductor surface 3 MAY 2002 VOL 296 SCIENCE www.sciencemag.org P892-895

  12. Phage display to align the nanoparticles Repeat the bioselection Semiconductor surface 3 MAY 2002 VOL 296 SCIENCE www.sciencemag.org P892-895

  13. Phage display to align the nanoparticles Amplification Semiconductor surface 3 MAY 2002 VOL 296 SCIENCE www.sciencemag.org P892-895

  14. CONTINUED The bacteriophage were coupled with ZnS solution precursors andspontaneously evolved a self-supporting hybrid film material that wasordered at the nanoscale and at the micrometer scale into ~72-micrometer domains, which were continuous over a centimeter lengthscale.

  15. Phage display to align the nanoparticles Liquid crystal formation 3 MAY 2002 VOL 296 SCIENCE www.sciencemag.org P892-895

  16. Phage display to align the nanoparticles ZnS nucleation and binding Zn2+ + S2- ZnS 3 MAY 2002 VOL 296 SCIENCE www.sciencemag.org P892-895

  17. Optical Microscope Electron Microscope AFM Phage display to align the nanoparticles

  18. Application: Self-assembly of semiconductor chips Phage display to align the nanoparticles 3 MAY 2002 VOL 296 SCIENCE www.sciencemag.org P892-895

  19. Self-Assembling DNA Nanostructures Double-stranded DNA shown in the standard, righthanded, B-form double helix with four base ssDNA sticky-ends appended to the 3' ends of both strands. Strand backbones are highlighted with colored ribbons; bases (light gray) are viewed edgewise and can be seen to point toward their hydrogen bonding partner on the opposite strand. Ref: http://www.cs.duke.edu/education/courses/spring02/cps296.5/papers/labean_CBGI_chapter.pdf

  20. Targeted metallization of a complex DNA superstructure. a). Filaments of DNA lattice constructed from Targeted metallization of a complex DNA superstructure. a). Filaments of DNA lattice constructed with the addition of two thiol (-SH) groups to the dsDNA stem protruding from one side and one amino group on the end of the dsDNA stem protruding from the other side of the tile. It appears that the thiol sulfurs associate with one another causing the lattice to curve and form tubes of quite uniform diameter. b). Same as a) with addition of 1.4 nm nanogold targeted to the amino groups on the protruding DNA stem. c). Same as b) with addition of 2 minute development with a silver enhancement procedure which deposits silver upon existing bound gold particles. d). Same as b) but with 5 minute silver enhancement. Progressive buildup of metal atoms is observed, with perhaps a few more minutes of silver binding required to form a complete, conductive wire. Note that these DNA filaments still have sticky-ends available at both ends which can be used for orienting the entire filament prior to metal binding. Ref: Introduction to Self-Assembling DNA Nanostructures for Computation and Nanofabrication. Thomas H. LaBean Department of Computer Science Duke University http://www.cs.duke.edu/education/courses/spring02/cps296.5/papers/labean_CBGI_chapter.pdf

  21. CHAPERONINS FOR THE FABRICATION OF QUANTUM DOTS WITH NANOSCALE RESOLUTION What are chaperonins? Chaperonins are hollow double ring structures composed of protein subunits called heat shock proteins (HSP60s). Their predominant function is thought to be facilitating protein folding inside cells, he said. Proteins fold to change shape at the molecular level, which allows them to carry out specific life processes. Reference: "Ordered Nanoparticle Arrays Formed on Engineered Chaperonin Protein Templates," Nature Materials, November 25, 2002

  22. Researchers from NASA, the SETI Institute and Argonne National Laboratory have genetically modified a bacteria that lives in geothermal hot springs in order to make a microscopic scaffolding that produces a high-tech material. The researchers used a protein from Sulfolobus shibatae, an extremophile organism that can grow in temperatures as hot as 85 degrees Celsius. The bacteria's ability to withstand high temperatures means the protein, called chaperonin, is particularly stable Reference: "Ordered Nanoparticle Arrays Formed on Engineered Chaperonin Protein Templates," Nature Materials, November 25, 2002

  23. STEP 1. The researchers used one of the three types of proteins that make up the chaperonin subunits. They altered the Sulfolobus genes to produce a protein with a slightly different structure, then inserted the altered genes into common E. coli bacteria, which manufactured large amounts of the modified protein. They heated the E. coli to 85 degrees Celsius to destroy the E. coli and its own proteins, leaving behind the engineered Sulfolobus protein. STEP 2. The researchers engineered two variants of the protein. To get one variant, they removed a portion of the gene that causes bits of protein to partially block the openings at the ends of the chaperonin. The partially blocked variant had an opening of 3 nanometers and the unblocked variant had an opening of 9 nanometers. Reference: "Ordered Nanoparticle Arrays Formed on Engineered Chaperonin Protein Templates," Nature Materials, November 25, 2002

  24. The red ring represents a genetically modified protein. The black and white microscope image shows an array of the rings. Cysteine residues Reference: "Ordered Nanoparticle Arrays Formed on Engineered Chaperonin Protein Templates," Nature Materials, November 25, 2002

  25. STEP 3. The genetic code of both variants was mutated to produce cysteine residues on the chaperonins' openings. Cysteine, an amino acid, acts like glue to bind a bit of gold or zinc to each opening like sticking a ball to the end of a tube. STEP 4. The researchers crystallized the altered chaperonins into flat, hexagonally packed templates. These chaperonin crystals arranged 5-nanometer quantum dots into arrays when the researchers used the variant with the 3-nanometer opening, while the the 9-nanometer chaperonin arranged 10-nanometer quantum dots. Reference: "Ordered Nanoparticle Arrays Formed on Engineered Chaperonin Protein Templates," Nature Materials, November 25, 2002

  26. This graphic shows the protein rings filled with microscopic specks of gold. The protein rings assemble themselves into arrays and capture gold specks, forming regularly spaced arrangements of the gold. Reference: "Ordered Nanoparticle Arrays Formed on Engineered Chaperonin Protein Templates," Nature Materials, November 25, 2002

  27. QUESTIONS?

  28. GLOSSARY Phage displayPhage display vectors express the inserted DNA as a protein at a prominent position on their capsid. This allows capture and isolation of recombinant phage clones by immobilised interacting proteins (eg. antibodies). This screening method is called biopanning.Oligonucleotide A short nucleic acid molecule; normally refers to molecules between 5 and 200 nucleotide residues(bases) long.5' or 3' endThe nucleoside residues which form nucleic acids are joined by phosphodiester linkages between the 3' C atom of one ribose moiety and the 5' C atom of the next. Therefore each strand of DNA or RNA has a free 3' C at one end and a free 5' C at the other. The free 3' C normally carries a - OH group, and the 5' C a phosphate group

  29. QUESTIONS FOR YOU 1. What is Biopanning? 2. What are chaperones? Why were many of them originally called heat shock proteins?

  30. Figure 2: DNA strands are antiparallel (the 5' end of one strand pairs with the 3' end of the opposite strand).

  31. Nucleotide = Base + Sugar + Phosphate (1, 2, or 3) Figure 1: Nucleotide structure

  32. FEEDBACK ----------------------------------------------- Date: June 06,2003 Presenter’s Name: Viswaprakash Nanduri Name of student turning in the form: ------------------------------ From 1 to 10 (ten being the best) How do you grade the materials presented? ------------------- Were complete references given for each slide? ------------- How well is the presentation understandable? ---------------- How are the glossary, questions and problems Presented? ---------------------

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