Imaging Nucleic Acids with the AFM. W Travis Johnson PhD Agilent Technologies Nanomeasurements Division

Transcription

1 Imaging Nucleic Acids with the AFM W Travis Johnson PhD Agilent Technologies Nanomeasurements Division

2 Structure of DNA A T G C Standard Watson-Crick A-T & G-C base pairs in B-DNA DNA double helix composed of A-T and C-G base pairs - attached to negatively charged sugar-phosphate backbone.

3 The DNA Double Helix Forces Which Stabilize DNA Exclusion of Water excluded from helix core entropy allows specific H-bonds between strands Electronic Effects π π stacking interactions nearest neighbor effects (sequence NOT composition) nm Positive Ions (metals) phosphate interactions (charge neutralization) Na, Zn, Mg, etc Specific H-Bonds G-C (3) A-T (2)

4 A C B D AFM Basic Configuration Z Y X Reconstruction AFM Topography Image

5 Contact Mode & AC Mode AFM Contact Mode AFM Dynamic in x and y. Tip is in contact or near contact with the surface. Small vertical force, but the probe dragged over the surface exerting lateral force. Weakly bound or soft samples move easily. Low lateral resolution. AC Mode AFM Dynamic in x, y, and z. Intermittent contact. Impact is predominately vertical, therefore large vertical force, but small lateral force. Soft surfaces are stiffened by viscoelastic response Higher lateral resolution.

6 Sample-Surface Interactions vs Probe-Sample Forces AFM probe exerts lateral forces on sample Sample-Surface interactions must be stronger than Probe-Sample forces

7 Sample-Surface Interactions & Probe-Sample Forces Electrostatic/Ionic/Charge-Charge Interactions H Bonds: RO.. H RS.. H R 2 N.. H Attractive Ionic Repulsive Ionic Hydrophobic Interactions VDW Interactions Hydrophobic Attractive VDW Repulsive VDW

8 Sample-Surface Interactions & Probe-Sample Forces Hydrodynamic & Fluid Forces As tip approaches surface, viscosity of confined liquid between tip and sample increases In AC Mode, the oscillating probe squeezes fluid in and out between the probe tip and surface

9 Attaching DNA to Mica: Polylysine +OH 2 +OH 2 +OH 2 +OH 2 +OH 2 -O -O -O -O -O RO Si O Si O Si O Si O Si OR RO Si O Si O Si O Si O Si OR pka=2.3 pka=6.8 silanol groups deprotonate in aqueous solutions to give negatively charged surfaces Mica DNA/RNA (PO 4 -) Polylysine

10 Amination of Mica Using Aminosilanes a. APTES: an alkoxyaminosilane toluene or acetone (contains H 2 O) b. Results in solution: a. Desired bond formation with substrate c. b. Hydrolysis c. Condensation polymerization and partial condensation on surface => makes mica rough

11 Surface Roughness of MICA-APTES: Comparison of Solution Phase vs Vapor Phase Deposition µm nm µm nm µm Cleaved Mica + APTES toluene; rms 5 nm µm Cleaved Mica + APTES vapor phase; rms.18 nm µm nm µm Freshly Cleaved Mica rms.16 nm

12 Vapor Phase Amination of Mica APTES aminopropyltriethoxysilane Aminated substrate Atomically flat/smooth APTES argon Lohr et al Methods 41 (27)

13 Attaching DNA to Mica: Aminosilane (APTES) APTES vapor DNA/RNA (PO 4 -) Buffer ph 7.4 H 3 N H 3 N H 3 N H 3 N H 3 N Si O Si O Si O Si RO O Si OR O O O O O Si O Si O Si O Si RO O Si OR

14 DNA on APTES-Mica µm nm µm Length = 1.5 µm Pt = 1.72 nm Scale = 1.72 nm µm

15 Dull Tip on AFM Probe Line Broadening n m µm µm L e n g th = 1.5 µ m P t = n m S ca l e = n m µm

16 Immobilizing DNA on Mica with Multivalent Metals M ++ DNA/RNA (PO 4 -) µm µm nm Length = 1 µm Pt = 2.6 nm Scale = 2.6 nm Negatively charged molecules (DNA, RNA, viruses, etc) immobilized on mica using divalent cations (Mg++, Ni++, Co++, Zn++, Cu++) µm

17 Timing of the Addition of the Metal Ions: DNA-M ++ -DNA vs DNA-M ++ -Mica NiCl 2 added before DNA spotted on Mica DNA strands bind to each other µm µm µm µm

18 DNA Minicircles (168 bp) MgCl 2 +ZnBr 2 Han, PNAS, Vol. 94, pp

19 Viral Capsids and RNA on Ni ++ - Mica: HRV2/RNA ph= hours 3 nm 3 nm Kienberger et al., J. Virol. 78 (24) 323

20 DNA Origami (6x9nm) + 5nm Au Nanoparticles (AC Mode in TAE/MgCl 2 Buffer) 2 nm Image Courtesy of Prof Hao Yan, Arizona St University

21 AAC Mode Image of DNA on Ni ++ -Mica: Ambient Conditions/Wash with Water Before Imaging µm µm

22 AAC AFM Image of Agilent DNA Microarray Probe Element µm µm nm nm Length =43.1 µm Pt = 4.89 nm Scale = 4.89 nm µm

23 AAC AFM Image of Agilent DNA Microarray Probe Element nm

24 Area Between Probe Elements (1x1 um)

25 Thank You! Questions? W Travis Johnson w-travis_johnson@agilent.com

26 END