Stretching DNA as a template for molecular construction

Size: px

Start display at page:

Download "Stretching DNA as a template for molecular construction"

Allyson Mosley
6 years ago
Views:

title Workshop " DNA-Based Molecular Electronics" Jena, May 13-15, 2004 Stretching DNA as a template for molecular construction Masao Washizu 1, Yuji Kimura 1, Takuya Kobayashi 1, Osamu Kurosawa

1 title Workshop " DNA-Based Molecular Electronics" Jena, May 13-15, 2004 Stretching DNA as a template for molecular construction Masao Washizu 1, Yuji Kimura 1, Takuya Kobayashi 1, Osamu Kurosawa 1,2 Sayoko Matsumoto 3 and Takayoshi Mamine 3 1 Department of Mechanical Engineering, The University of Tokyo 2 Advance Co. 3 Sony Corporation Contact: washizu@washizu.t.u-tokyo.ac.jp

2 Stretching DNA as a template for molecular construction stretched single-stranded ss-dna Construction of Ordered Molecular Units resolution = 1 base = 0.34nm Potential application for 1) Molecular electronics devices 2) Functional molecular (enzymatic) units

3 Application for molecular devices? Alignment of a device electrode molecular diode conductor conductor template single-stranded DNA electrode Or more complex molecular circuits GNDCHOHOCCODATAREADWRITECHOHOCCOSTORAGE NO CHOHOCCOGATESOURCEDRAINQUANTUM DOT

4 Requirements Straight template on a predetermined location 1) To avoid entanglement or short circuit. 2) To facilitate evaluation. 3) To avoid back-coiling that hampers molecuar interaction. Very high yield of binding η For a device consisting of N components η total = η N N = 10, η = 90% η total = 0.35 N = 100, η = 90% η total = N = 100, η = 99% η total = 0.37

5 FISH (Fluorescence In Situ Hybridization) chromosome probe fluorescence dye oligo nucleotide The sequence is there!! A kind of molecular construction, but.

6 FISH is essentially a low-yield process The probe must break into the already existing base-pairs of double-stranded (ds-) DNA. Process Strand separation (denaturing) Mix with the probe and anneal Must compete with back-pairing also.

7 In order to achieve a high-yield binding 1) Bases must be exposed to allow free interaction with foreign molecules (i.e. the probe). 2) The template must be held straight to avoid back-folding.

8 Electrostatic Stretch-and-Positioning of double-stranded DNA a) b) DNA Electrode Electrode DNA takes random conformation in water. d) c) DNA polarizes under 1MV/m 1MHz field. Move towards electrodes until one end touches. Permanently anchored on aluminum electrode. DNA stretched by electrostatic force

9 Electrostatic Stretching of DNA fluorescence image dye = DAPI λ-dna 48kb =16 µm 80 µm

10 DNA high-wire system floating-potential electrodes (FPE) energization electrode bird's eye view cover slip spacer photo glass substrate DNA DNA 20 µm 1-3 µm ~16 µm gap DNA is stretched and held at ends ; no hindrance for the interaction with foreign molecules. electrode

11 Stretch-and-positioning of double-stranded DNA in the high-wire system electrode electrode electrode electrode electrode 40 µm

12 Why ss-dna more difficult to stretch? ds-dna ss-dna Internal base-pairing Hydrophobic folding Lp bp? (flexible) Stiffness due to 1) paired structure 2) charged backbone Persistent length Lp 300 bp Mechanical integrity: single backbone defect results in breakage No good add-on fluorescence probes like intercalators for ds-dna

13 Direct observation of ss-dna stretching Fluorescence-label ss-dna itself Primer 1 (with biotin) template DNA Primer 2 (no biotin) PCR with fluorescein-12- dutp + dntp Immobilize biotin end Recover one strand product To prevent ds formation, one of the strands must be recovered exclusively. Purity checked by ss-cutting S1 Nuclease.

14 Electrostatic Stretching of Single-Stranded DNA 10kbp ds-dna 10kb ds-dna

Stretched length as a function of applied field Stretched Length L [mm] 10kbp ds-dna Stretched Length L [mm] 10kb ss-dna Field Strength E [MVpeak / m] Field Strength E [MVpeak / m] 1) ds-dna

15 Stretched length as a function of applied field Stretched Length L [mm] 10kbp ds-dna Stretched Length L [mm] 10kb ss-dna Field Strength E [MVpeak / m] Field Strength E [MVpeak / m] 1) ds-dna stretched almost to the full length (= 10 kb x 0.34 nm/base), while the stretched length of ss-dna is about 2/3. 2) ss-dna requires higher field than ds. 3) Hysterisis observed in ss-dna. Difficult to stretch, but once stretched, rather easy to maintain stretched conformation.

16 ss-dna bridging over the electrode gap Probe binding experiment now underway

17 Base-exposed stretched double-stranded DNA as an alternative use as the template ss-dna : mechanically weak If bases can be exposed from stretched ds-dna Recombination protein should have such function. It will have same effect as stretched ss- DNA.

18 Homologous recombination Recombination protein such as RecA or Rad51 uses intact homologous DNA to repair molecular damage. In the process, it opens up ds-dna and accesses the bases inside. sequence-probing function ds-dna breakage removal of damage RecA binding homology search DNA repair binding to the sequences of interest The structure of RecA

19 Preparation of fluorescence-labeled DNA YO-PRO-1 "DNA probe" RecA ssdna (~500 base) dsdna (~18 kbp) RecA-binding ss-dna Fluorescence-labeled by YO-PRO-1

20 Fluorescence image of the DNA probe binding with DNA DNA highwires DNA probe electrode electrode electrode Solid surface between the electrodes is out of focus. view DNA length = 16 µm

21 Sliding of DNA probe along the target DNA Among the DNA probes that bound on DNA, only a few percent of such probes slid along the target DNA. Most probes once bound moved only slightly.

22 Binding position of the DNA probe experimental result homologous site Frequency The histogram has peaks at near the homologous site, and dips on both sides. Also seen is the decay at both extremes of the ds-dna bridge. The width of the dip may indicate the diffusion length of the probe along DNA The position of the DNA probe along the target DNA (%) 100% = length of target DNA = 16 µm

23 Conclusions For the use of DNA as the template of molecular construction, Single-stranded DNA high-wire system is developed. The stretched length of ss-dna is observed to be about 2/3 of that of ds. Probe binding experiment underway. RecA in combination with ds-dna high-wire may be an alternative. Binding to homologous sequence observed, but other peaks as well, possibly because of too strong interaction (too many RecA?)

24 Acknowledgments Mr. Osamu Kurosawa (Advance Co.) Dr. Hiroyuki Kabata (Kyoto Univ.) Dr. Hidehiro Oana (The Univ. of Tokyo) BRAIN (Seiken-Kiko), R&D Program for New Bio-Industry Initiatives Ministry of Education, Kakenhi No. A Advance Co. Sony Corporation

Bootcamp: Molecular Biology Techniques and Interpretation

Bootcamp: Molecular Biology Techniques and Interpretation Bi8 Winter 2016 Today s outline Detecting and quantifying nucleic acids and proteins: Basic nucleic acid properties Hybridization PCR and Designing