Weight Encoding Methods in DNA Based Perceptron

1. Weight Encoding Methods in DNA Based Perceptron 2004. 7. 6 임희웅

2. Contents • Weight Encoding Criteria • Methods • Internal fluorophore • Fluorescence resonance energy transfer • Competitive hybridization of labeled and unlabeled probe • Materials

3. Weight Encoding Criteria • Output • Fluorescence Signal • Proportional to • Concentration of corresponding mRNA (xi) • Corresponding of weight value (ti) Assumption: Final signal (output) is the sum of each output signal Outputi

4. Internal Fluorophore Absolute Weight Value High Low (+) Fluorophore 1 Sign (-) Fluorophore 2 More fluorophores Less fluorophores

5. Fluorescence Resonance Energy Transfer • Fluorescence Resonance Energy Transfer • Transfer of the excited state energy from the initially excited donor (D) to an acceptor (A) • Distance-dependent interaction between donor and acceptor without emission of a photon

7. Quencher • Small weight  short probe length  weak signalLarge weight  long probe length  strong signal Absolute Weight Value High Low (+) Fluorophore 1 Sign (-) Fluorophore 2 Longer probe Shorter probe

8. FRET Efficiency • Förster Distance (R0) • A distance where the efficiency is 50%.

9. In case of R0=50

10. DNA Helix Model for FRET Efficiency Clegg et al. PNAS 1993

11. Dietrich et al. Reviews in Molecular Biotechnology 1993

12. Our Dye-Quencher Pairs • How Many? • 2 pairs for (+) and (-) • Condition • Dark quencher • No background signal, high S/N ratio • Discrimination between (+) and (-) • Large difference in wavelength peak • Multiplexing • Small interference between two dyes. • How about using the pairs in RT-PCR? • Sensitivity • Efficient sensitivity in the concentration of experimental condition. • Enough Förster Distance (R0) • Active range of FRET corresponding to probe length • 3.3Å for one bp,

13. 6FAM cy5 Rox (Texas-red) Hex And BHQ series as dark quencher

14. More to Know • Fluorescence intensity vs. concentration • Multiplexing

15. Competitive hybridization of labeled and unlabeled probe • Small weight  low ratio of labeled probe  weak signalLarge weight  high ratio of labeled probe  strong signal Unlabeled Probe Labeled Probe Excess Probe Competitive hybridization mRNA

16. Sign Encoding with One Fluorophore probe Positive weight mRNA Negative weight High Conc. Low Conc.

17. Reference • Clegg et al. PNAS vol. 90, p 2994~2998, 1993 • Observing the helical geometry of double-stranded DNA in solution by fluorescence resonance energy transfer • Dietrich et al. Reviews in Molecular Biotechnology, vol. 82, p 221~231, 2002 • Fluorescence resonance energy transfer (FRET) and competing processes in donor-acceptor substituted DNA strands: a comparative study of ensemble and single-molecule data

18. Materials

19. Dual Labeled Probe (1) Bioneer http://www.bioneer.co.kr/biomall/mall_oligo.jsp

20. Dual Labeled Probe (2) 5’/3’ 1. FAM/BHQ-1 Fluorogenic Probe 2. HEX/BHQ-1 Fluorogenic Probe 3. TET/BHQ-1 Fluorogenic Probe 4. MAX/BHQ-1 Fluorogenic Probe 5. Cy5/BHQ-3 Fluorogenic Probe 6. Cy5/BHQ-2 Fluorogenic Probe 7. Cy3/BHQ-2 Fluorogenic Probe 8. TAMRA/BHQ-2 Fluorogenic Probe 9. ROX/BHQ-2 Fluorogenic Probe Synthegen http://www.synthegen.com/

21. Dual Labeled Probe (3) IDT http://www.idtdna.com/program/catalog/Dual_Labeled_Fluorescent_Probes.asp

25. Förster Distance (R0) HiLyte Biosciences, Inc.

26. http://www.nlv.ch/Molbiology/sites/Fluorescence4.htm

27. Dark Quenchers

29. http://www.biosearchtech.com/ This chart shows the absorption spectra of all three BHQ dyes (conjugated to poly-T 9-mers and normalized to the T9 absorbance at 260 nm) with the emission maximum of many commonly used reporter groups indicated.

30. Multiplexing Instrument