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DNA COMPLEXES ON A BIOSENSING SURFACE

Explore the role of DNA complexes in biosensing, including DNA hairpins, G-quadruplexes, DNA nanotechnology, and various DNA-based sensors and amplification techniques.

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DNA COMPLEXES ON A BIOSENSING SURFACE

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  1. DNA COMPLEXES ON A BIOSENSING SURFACE Luka Vanjur luka.vanjur@unimi.it

  2. DNA – themoleculeoflife • Deoxyribonucleicacid • Repositoryofgeneticinformation • Twolinearpolymers • Monomers – nucleotides • Doublestranded (ds) • Adenine (A), Cytosine (C), Guanine (G), Thymine (T) • Watson-Crick pairing: • A-T • G-C 5’ 3’ 0.34 nm H2N N H O O N N CH2 P O- O 3’ H H 5’ N 2 nm O- H H H H OH

  3. DNA – themoleculeoflife DNA hairpin 5’ • Single stranded (ss)DNA • One linearpolymer • Monomers - nucleotides • Intra-strandpairing • Hairpins • G-quadruplexes 0.67 nm G-quadruplex H2N N H O O N N CH2 P O- O 3’ H H N O- H H H 1 nm H OH

  4. DNA – themoleculebeyondlife • DNA nanotechnology • Structural – DNA origami • Functional – DNA nanomachines i. ii. AFM imageofvarioussymbolsproducedwithDNA origami [Weietal. Nature485, 623–626(2012)] iii. iv. DNA tweezers

  5. DNA on surface f -fraction of bound sites Solution • Surface vs. Solution: • Loweraffinityandslowerkinetics • Electrostatics • DNA ischarged (-Ne perchain) Surface Depletion zone

  6. [1] Seeman N.C., J TheorBiol.,21;99(2):237-47 (1982) [2] Rothemund P.W., Nature, 440(7082), 297-302 (2006) Twoschoolsof DNA design • Scaffold-basedapproach [2] • Tile-basedapproach [1] A T A T C G A T G G A A T A C C T T

  7. ReflectivePhantomInterface (RPI) • DNA microarray • Optical, multiplexed, label-free biosensor [4] • Verylowinitialreflectivity (R0~10-5) • RPI 2.0 • Fluorescence + RPI • Single-moleculeevents [4] F. Giavazzi, M. Salinaetal., PNAS 110,9350 (2013)

  8. DNA bush • Surfaceboundaptamer „web” • Bacteriadetection • Tile basedapproach • Responsivehydrogel

  9. Rolling CircleAmplification • Phi29 – DNA polymerase (enzyme) [5] • LongssDNA (40mer → ~1k bases) • Tandem repeats primer template dNTPs circular DNA Phi29 polymerasecartoon; top view (top) side view (down), PDB: 1IJG [5] Ali M.M. etal., Chem. Soc. Rev., 43, 3324-3341, (2014)

  10. HybridizationChainReaction • Signal amplification • Interactinghairpins • Initiallytrapped (kinetically) • Triggeredself-assembled DNA nanostructure H3 H2 free energy H1 T kinetictraps P vi. iii. v. i. ii. iv. reactioncoordinate

  11. miRNAdetection AuNP • Micro-RNA • ~20 nucleotides • Lowabundance • FunctionalizedAuNP • Signal amplification • DNA-directedimmobilization [6] AuNP miRNA [6] Niemeyer C.M., Anal Biochem., 268(1), 54-63, (1999)

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