Presentation on theme: "Bath and Turberfield review Toehold displacement Light and dark versions of same color = complements."— Presentation transcript:
Bath and Turberfield review Toehold displacement Light and dark versions of same color = complements
initiator acts as catalyst – speeds up rxn without being depleted fast catalyst slow topological barrier to hybridization relieved by catalyst strand (“=initiator”) Would a little heat remove barrier?
More complex version with multiple hybridization steps before initiator (I) is released Gel electrophoresis separates DNA species by size “leakage” hybridization => kinetic traps not absolute
AFM images of 3 and 4 arm products show expected species are formed
tweezers/actuator open unset strand closed set strand waste toe-holds DA and are fluor. dyes How to tell if it changes configuration? whose fluorescence changes with separation distance tutorial on fluorescence http://probes.invitrogen. com/resources/education/ tutorials/2Spectra/player. html
donor acceptor Excite at 575nm … If acceptor molecules are close to donors (r small), you get less emission at ~650nm and more at ~780nm than if r is large FRET = fraction of donor absorp. energy that is emitted by acceptor Typically, R 0 ~6nm r K r -6 from dipole-dipole int.
Related application makes sensor for tgt dna = molecular “beacon” Use non-fluorescent acceptor! http://www.molecular-beacons.com/ n base loop comple- mentary to part of tgt How long should n be to reduce quench (brighten) 33-fold? (hint: 10 bp one turn 3nm, ) tgt stem << n stem regions
“Exponential” version of toe-hold displacement: Each cycle produces new catalysts, so rate of hybridization accelerates until all DNAs have annealed Monitor rxn with FRET dye and quencher
Time to make threshhold amt. of product inversely [I] Like pcr, t c = -k ln[I] => exp. process But only true over 15x range Note spontaneous hybridization also exponential, which limits sensitivity to [I] >100pM ( 10 8 / l, so poor cf. to pcr)
More comparisons to pcr In exp. toe-hold displacement system, is the product produced in the absence of initiator (analyte) different from the product produced in its presence? In pcr, is product produced in the absence of template (analyte)? If “primer-dimer” is amplified in the absence of template, can one distinguish it from correct product (e.g. with beacon)?
Another “exponential” toe-hold system based on 1 melted stem-loop melting 2 others, each of which melts 2 others … AFM evidence for “dendrimers” But process limited by steric hindrance after a few levels
Bi-pedal walker on stem-loop track Fuel, B, has toehold c that displaces walker leg from A Bi-pedal walker has toehold a’ that melts A stem Lots of effort to make dna “machines” that move along a track, similar to natural biological motors (class 10)
If B displaces left leg first, walker moves 1 step forward if B displaces right leg first, walker is displaced off track Same at each subsequent step Walker may fall off, but it can’t go backwards, because sites where it has been are occupied by fuel This is “burnt bridge” forward bias mechanism
How to detect biased movement to right? Put green, red, blue fluors at sites 3, 4 and 5, quencher on walker, see if 3 quenched before 4 before 5
3 4 5 Controls change order of colors 1-leg walker 3 4 5shows little bias Note how small the effect is! Why does 1-leg walker ever get to position 4 or 5?
Think carefully about directional bias in these systems Here it comes from biased starting position – unlike biological motors where bias comes from molecular asymmetry Others have made walkers with biased directional walks based on differential release of R vs L legs, due to differential exposure of toe-hold sequences on L vs R legs (clever, but complicated, need to see picture!) No burnt bridges necessary, more like some natural biol. motors (if interested, see Turberfield et al Phys Rev Let 101:283101 (2008))
Aptamers – single-stranded NA that binds to a tgt molecule via non-base pairing interactions e.g. RNA segment that binds sugar or ATP, often part of control pathway to sense tgt and turn off transcription or translation NA sequence that binds particular tgt can be selected from huge “library” of different sequences
F, R = regions of particular seq. 20b long V = region of variable seq. made by adding mixture of A, C, T, and G at each position Library = collection of oligos with same beginning and end sequence but different “middles” 5’agccg…cgcat-nnnnn…nnnnn-gacgt…cagga F R V Make “library” of chemically synthesized oligonucleotides How to find aptamer that binds tgt molecule Z
Pour library over column with immobilized Z Wash away what doesn’t stick to Z Elute (e.g. with boiling water) what did stick PCR amplify eluate with F and R’ -> repeat… You may end up with a few oligonucleotide species (particular sequences) that bind Z tightly How many positions (N) should be variable, assuming you can process 30 g of oligo? (hint: how many oligo molecules if length is 20+20+N; how many variable positions to make this # of variants) Are you impressed with how many different oligos you can screen?
The same process was used to search for oligonucleotides with enzymatic activity, e.g. that could degrade RNA (RNA’se) Can make this aptamer specific for a particular sequence with “arms” that bind the tgt RNA’se aptamer 2 rna bases rest of oligo is dna targeting arms
not x on y y and not x x on off y and not x indicator not x indicator sensors with logical f n read-outs but how tight are controls? you can control the activity of modified versions of this RNA’se aptamer with other dna’s in ways akin to logical circuits can check function of control strands (x, y) by FRET or assay for cutting activity
Another use of dna enzyme – “walker” that eats its track Why does walker go in one direction in illustration? What happens to “directionality” if the cutter completely dissociates from anchors and reanneals in middle of track?
Variant walker with multiple-legs = “spider” 2-d tracks made with dna “origami”
Walker driven by sequential addition of toe-hold displacement and attaching strands Note you need complicated track with ordered series of anchors
Rotating “walker” based on clever use of 2 restriction enzymes, ligase, and ordered track of anchors You need to see details of cutting enzymes to understand how it works
Where does directionality come from? (This can be subtle!) Recall ligation does not necessarily restore restriction sites (1 st class, identical sticky ends from different enzymes), so it does not have to be reversible. Here ligation of some products is irreversible (e.g. B* with C). It then creates new sites, which allow the ligation products to be cut into new products (e.g. B, C*, whose religation/cutting happens to be reversible). Directionality comes from the irreversibility of some ligations and the pre-arranged order of the anchors. Directionality/irreversibility requires consumption of energy (Maxwell’s demon, 2 nd law of thermodynamics); here this comes from ligation, which hydrolyzes (splits PP off of) ATP.
Comments about Tuberfield et al review Nice description of collection of concepts with numerous cited realizations (good list of potential papers for student presentations) But little critical evaluation – e.g. what is background signal when target sequence is not present, how big is difference between “on” and “off” signals, how efficient are “DNA enzymes” in cutting, how sensitive are detectors? Advice: appreciate ideas but critically evaluate examples
Summary Clever uses of toe-hold displacement, catalytic oligos, restriction site calisthenics -> catalytic displacement rxns exponential displacement schemes biased molecular walkers But compared to natural biological machines, these devices are terribly crude. Think of DNA polymerase as a motor, >100 bp/s, processive (stays on track) for 1000’s of bp, doesn’t go backwards except to correct errors. So far man-made motors are >10 4 times slower, weakly processive, weak directional bias
Be creative in thinking about new things you might make with DNA – the field is young, not many people are involved, many ideas will not yet have been thought of or tried
Next week – novel use of DNA to purify carbon nanotube species CNT = single layer sheet of carbon rolled into tube; tubes have different properties (metallic, semiconducting) depending on diam. and twist; lots of promising applications but problems purifying different species
CNTs very hydrophobic (just C), aggregate in water Can “solubilize” with soap = molecule with hydrophobic end and hydrophilic end ssDNA acts as “soap”! – bases “stack” on CNTs, phosphates are hydrophilic
Next week’s paper shows different CNT species can be purified using different DNA oligonucleotides + ion-exchange chromatography Mechanism not understood – postulated to involve novel DNA structures on CNT surface method potentially of great practical significance amazing all the things one can do with DNA! Read – Nature paper by Tu, including Supplement, and news and views commentary (N&V)