Prof. Kotlyar Alexander DNA nanolab


                                        AFM image of DNA molecules attached to Avidin  (bright spots) aligned on the surface                                          

Affiliation:           Biochemistry & Molecular Biology, The George S.Wise Faculty of Life Sciences
Tel:                     (972)-3-6407138
Fax:                    (972)-3-6406834
Email:                 s2shak@tau.ac.il
Postal Address: Dept. Biochemistry & Molecular Biology, Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel




DNA – based Nano Wires


DNA is a fascinating soft material that naturally expresses two of the three main features required from molecular nanoelectronic components, namely recognition and specific structuring (sequence, length). The third additional property that is needed in order to implement DNA-derivatives for electrical device applications is conductivity. The central objective of this multidisciplinary project is development of DNA-based conductive nanowires and nanodevices for nanoelectronics. 

The first goal of the work is synthesis of double-stranded [1], triple–stranded [2]  and G4-DNA [3, 4] nanowires was conducted using DNA Polymerase. We have demonstrated that this enzyme is capable of extending short double-stranded (ds) blunt-ended oligonucleotides composed of only several base pairs yielding very long (up to several microns) ds polymers [1].   


Extension of short blunt-ended oligonucleotide composed of 12dG and 12 dC bases, 12(dG)-12(dC), to yield long (thousand of GC base pairs) DNA (see [1])


We have demonstrated guanine quadruplex (G4-DNA) [4] that composed of four G-strands have higher electrical polarizability [6and conductance [7] versus natural double-stranded DNA. These G4-nanowires will be assembled into complex two-dimensional and three-dimensional DNA architectures and integrate functional units along with other molecular electronic components yielding interconnected networks, DNA-based nano-devices and nano-circuits.  This will lead us to realization of the main goal of this research i.e. the creation, by self-assembly, DNA-based nano-scale transistors and devices and that can be used to build computer chips and form the basis of a new type of information processing architecture. 

References

[1] A. Kotlyar, N. Borovok, T. Molotsky, L. Fadeev, M. Gozin "In Vitro synthesis of uniform Poly(dG)-Poly(dC) by Klenow exo-fragment of Polymerase I.", Nucleic Acid Research 33, 525 (2005);
[2] A. Kotlyar, N. Borovok, T. Molotsky, D. Klinov, B. Dwir, E.Kapon "Synthesis of novel poly(dG)-poly(dG)-poly(dC) triplex structure by Klenow Exo- fragment of DNA Polymerase I " Nucleic Acid Research 33, 6515 (2005);
[3] A. Kotlyar, N. Borovok, T. Molotsky, H. Cohen, E. Shapir, D. Porath "Novel Long Monomolecular G4-DNA Nanowires", Adv. Mater. 17, 1901 (2005);
[4]  N. Borovok, N, Iram, D. Zikich, J. Ghabboun, G. Livshits D. Porath, A. Kotlyar  "Assembling of G-strands into novel tetra-molecular parallel G4-DNA nanostructures using avidin-biotin recognition" Nucleic Acid Research 36, 5050 (2008);
[5] I. Lubitz, N. Borovok, A.B. Kotlyar "Interaction of monomolecular G4-DNA nanowires with TMPyP: evidence for intercalation" Biochemistry, 46, 12925 (2007):
[6] G.I. Livshits, J. Ghabboun, N. Borovok, A.B. Kotlyar and D. Porath  Comparative Electrostatic Force Microscopy of Tetraand Intramolecular G4DNA (Adv. Mater. 29/2014) Advanced Materials 26 , 5067 (2014);
[7] G.I. Livshits, A. Stern, D. Rotem, N. Borovok, G. Eidelshtein, A. Migliore, E. Penzo, S. J. Wind, R. Di Felice, S. S. Skourtis, J. C. Cuevas, L. Gurevich, A. B. Kotlyar and D. Porath "Long-range charge transport in single G4-DNA molecules." Nature Nanotechnology 9, 1040-1046 (2014).
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Biomedical Applications of Plasmonic Nanostructures

Due to their attractive electronic, optical, and thermal properties noble metal nanoparticles have attracted huge interest of chemist, physicists and biologists. The nanometric size, unique optical, electrical and chemical properties led to a broad range of the metal particles application in various research fields, including nanophotonics, nanoelectronics, optoelectronics, nanomedicine and others. In particular, the particles have been rather successfully used in therapy and diagnostics of cancer. 
The main goal of this research avenue is eradication of cancer cells using nanoparticles coated with proteins that capable of specific interaction with receptors that are overexpressed in cancer cells. One of these proteins is DARPin _9-29, an ankyril repeat protein that was designed to specifically target human epidermal growth factor receptor 2 (HER2) which is overexpressed in breast cancer. We have demonstrated that interaction of spherical gold nanoparticles (GNPs) with DARPin _9-29 yields very stable GNP-DARPin conjugates that do not aggregate at physiological salt concentrations and interact specifically with the surface of SK-BR-3 cells that overexpress receptor HER2 and are efficiently transferred into them by endocytosis [Bioconjugate Chemistry 2017 28 (10), 2569-2574]. Moreover, we have demonstrated that illumination of the DARPin-GNP-treated SK-BR-3 cells by red light (633 nm) leads to significant cell death; CHO cells, lacking the receptor, treated with the conjugate were not affected by the illumination. Specific eradication of cancer cells using GNP-DARPin conjugates pave the way to developing novel photodynamic approaches for specific treatment of cancer.
 



Specific interaction of DARPin-FITC-GNPs and DARPin-mCherry-GNPs conjugates with HER2 receptors overexpressed on the surface of SKBR-3 cells (A, B).  Confocal fluorescent images of the cells were acquired at excitation wavelengths of 488 (A) or 561 nm (B). Superimposed images of the cells in blue-green and blue-red fluorescence channels are presented in panels A] and B respectively. Nuclei were stained with Hoechst 33342. 
Schematic drawing of the conjugates between GNPs and Fluorescein-labeled DARPin (left bottom panel) and  DARPin-mCherry (right bottom panel).