Molecular ViewNanomaterialsStickyendsDNAFilmMgionsMicaLayer_ _ _ _ _ _ _ _+ + + + + + + ++_+ + + + + +Si _ Si _ Si _ Si _ Si _ Si _ SiO O O O O OO+_O
Designing theOligonucleotidesHairpins: DNA baseson one strand bindto itselfDimers: bases from twostrands of DNA bind atunfavorable locationsAreas of assemblydue tocomplementarybase pairs on samestrandStrand 2Strand 1Areas of assembly due tocomplementary base pairs at varioussites on separate strands of DNAThese cause interruptions or defects in the filmformations
2D Film AssemblySpacersStrand 1Each section of the oligosare 11 bases eachA D A DG-A-G-G-C-T-G-C-C-A-GC-T-C-C-G-A-C-G-G-T-CStrand 2Strand 3b d b dB C B C2 3Strand 4a c a c14Strands close uponthemselves
Assembly of 2D DNA Ribbons with “Triplet”TechniqueAssemble TripletsSeparatelyTriplet 1 Triplet 2Triplet 1 Triplet 2 Triplet 3 Triplet 4
Agarose Gel Electrophoresis• Will separate the structures that formedaccording to size• Higher molecular weight structures stay closer tothe wells• Lower molecular weight structures migrateacross the gelOligonucleotidemixed withbufferWellsMotion ofoligonucleotidesPositiveElectrodeNegativeElectrodeAgarose Gel
Oligonucleotide Assembly:Without Triplet TechniqueWellsMarkerDNAHigherMolecularWeightAgaroseGelLowerMolecularWeightMixtures prepared with four types of oligonucleotide strands per sample.Concentration of magnesium chloride varied for samples 1-4. Not manyhigh molecular weight compounds formed.
Using Oligonucleotides Triplets:to Prevent DNA BundlesHigherMolecularWeightWellsMarkerDNAHigherMolecularWeightLowerMolecularWeightAgaroseGelLowerMolecularWeightPicture 1: Oligonucleotidesassembled with three strands persolution- triplets 1,2,3 & 4.Picture 2: 10 microL of each ofthe four triplets mixed togetherthree times to assemble filmheatedat 37º, 47º, & 57ºC.
In the Immediate Future•Design a new set of oligonucleotides that are morespecific in their interactions•Assemble the oligonucleotides on a mica crystalsurfaceAcknowledgements•Helen Hansma and Emin Oroudjev•INSET
To Prevent Hairpins andDimersDesired interactions havehighest melting point usuallybetween 46ºC and 58ºCHairpin and dimerformations have amelting point between36ºC and 42ºCHeat the oligonucleotides up to 98ºC and let cool for an hour and ahalf. This allows the correct interactions between the strands to occurwhile the hairpin and dimer interactions form and then melt again.
The Importance of SpacersOligonucleotide StrandA-T-A-T-A-T11 bases with C, G, T, & AAdenine to Thymine formthe weakest bonds, sofirst to break•How long should they be?•What bases should they be composed of?•Should they interact with a spacer on a different strand?
Agarose Gel Electrophoresis• Will separate the structures that formedaccording to size• Higher molecular weight structures stay closer tothe gel• Lower molecular weight structures migrateacross the gelOligonucleotidemixture withbufferWellsPositiveElectrodeNegativeElectrodeAgarose Gel