DNA Nanotechnology Class Notes
Skewed TX motif
2D array is held together by double cohesion and forms nicely. Also failed to produce diffracting 3D crystal (x-ray crystallography). But double cohesion is potent method for producing cohesion in structural DNA nanotech. increases strength of DNA intermolecular interactions
Triplex DNA
in addition to double helical motif, there are other helical motifs that have been characterized. earliest of these was DNA triplex: pyrimidine-purine-pyrimidine structure formed. Without disrupting double helix, it's possible to address specifically designed locations within assembly by adding TFO (triplex-forming oligonucleotide) Can tether both small molecules and macro molecules to DNA constructs by use of triplex associations. can add triplex DNA to a DNA tensegrity triangle crystals as shown in figure
Four Arm Junction Arrays
it's immobile not actually a plus sign but more accurately a 60 degree angle. can add sticky ends to form center diagram. once self assembled, AFM will show the big spacing not the small spacing but you can measure the angles between domains. this is in regards to the bottom diagram.
3-turn and 4-turn tensegrity triangle
not significantly different from lattics designed from 2-turn tensegrity triangles. volumes are about 370 nm and 1000 nm respectively with resolutions ~6.5A and 10A respectively
6HB
Shih laboratory has made a 6HB about 1 micron long by combining two 6HB motifs designed from DNA origami. The purpose of these 6HB arrays is to produce long rods that can share a solution with membrane proteins without sticking to them. When the proteins collide with the long 6HB origami units, their rotational trajectories are slightly perturbed, leading to their partial align- ment. This partial alignment can be used in NMR experiments to establish the 3D structures of the proteins. if we consider DNA double helix to be 10.5 fold, then seven nucleotides corresponds to 2/3 of a turn, and 14 nucleotides corresponds to 4/3 of a turn. thus placing DX molecules around a cyclic path with connections every 7 or 14 nucleotides leads to 6HB motif
ZDNA promoting conditions
So why is it that yellow forms but blue and red doesn't? Yellow has the correct sequence (CG)n where n is ~10. the promoting condition is 10 milimolar cobalt hexamine. How do we know it formed? We can use a fret pair signal to detect the difference. on the top the pair is close together and down below it's far apart. the further the distance, the weaker the fret signal. ZDNA is on bottom You often find this in a transition state and this device is fine if you wanna just turn it over and turn it back. But what if you want a bunch of devices and want to be able to address them individually. Then, you need something that is sequence dependent.
Automobile Assembly Line
3 key components: worker, conveyor, factory worker= PX-JX2 cassette in various states inter-station conveyor = trigonal walker Framework = DNA origami - Here you are working with phentomoles of origami in micro liters, so you're working with nano molar chemistry but because you're working with such small quantities- you need a way to detect product that works on small quantities. so what you're going to add to growing construct is gold nanoparticles.
Compare
7.23A is analogous to 7.28Aand shows organization of the two molecules within the crystal Green and red molecules represent two different sepecies of tensegrity triangles- you can readily see from alternation of red and green molecules that each species of triangle is only bound to the other species. 7.23B is analogus to 7.28B and emphasizes cavities within crystal
XOR Operation
A cumulative XOR assembly is an instructive example. a. The XOR operation is an XOR logic gate- truth table at the left if the inputs are the same (0 and 0 or 1 and 1), then the output is 0. If they are different (0 and 1), the output is 1 This operation is just addition that lacks a carry bit. b. cumulative XOR, just using output from one XOR operation as one of the inputs to another XOR operation.
G Wire
A long purely parallel structure has been assembled into a G-wire. Its charge conduction properties are being explored
AB Coloring System
A macroscopic property has been programmed using a microscopic chemical attachment. the triangles themselves dont associate with themselves but only each other. if you add dye to A or to B or to both, there will be color in the AB system
Autonomous Walker Part 1
A- initial position of the walker. strand is linked 5'5' (the two circles) in the middle and its 3' ends are paired with two right strands of the T1 (LO (leg odd)) and T2 (LE (leg even)) stem loops which are openedT3 and T4 are intact and solution contains fuel f1 and F2. Sidewalk- long DX molecule B- special codes for colored region C- burnt-bridges (used up track) reaction whereby the two fuel molecules bind successively to stem loops and powering walker to the right Step size is larger here than in inchworm ** System is powered by two different fuel molecules that alternate in supplying the increased amount of base pairing that powers the device at every step Once you go forward, you can't go back
Adelman Experiment
Solved Hamiltonian Path problem using DNA molecules. Hamiltonian path problem is optimization of a route thorough number of cities in a teritory. If there are 10 cities, the number of possible answers is 10! or about 11 million. much harder if therre are 100 cities, then the possible amount of answers is proportional to 100! ***The notion behind molecular computing is that the huge multiplicity of molecules in a pot can be treated in parallel, rather than sequentially. This provides vast acceleration in calculation speed! Hamiltonian problem: 7 cities: Quito, New York, Paris, Johannesburg, Melbourne, Ulan Bator, and Shanghai.find a route from an origin city to a target city that goes through all of these cities once.
DNA Bricks
idea: large number of different individual strands can be assembled into a molecular canvas. . the complex shapes are produced by selecting the desired strands from the canvas, and then including only those strands in the final construct. *if a strand is on a border, then a second version of it, designed not to interact with other strands, will be used. *Canvas is the base rather than scaffold. Only have staples not scaffold. remove key strands from canvas
Coloring Crystals 2
nick in strand of tensegrity triangle and extend strand from the 3' end and it's got this red sticky end on its if we look in the lower left corner we can see because we only have dna we can see that we have another clear crystal. Add strand to crystal and we see that the crystal gets its color change because the strand itself has CY3 (red) so the crystal becomes dyed red. when you add CY5, it displaces CY3 and the CY3 goes into solution but the CY5 is much more intense this was an asymmetric triangle. extension (is colored) and it binds to central strand and colors the crystal. so it's an extra strand that binds to the central strand.
PNA
nucleic acid molecule with peptide backbone. most common: 6 bonds on its backbone unlike the 3-bond peptide backbone found in proteins. it has a base as a side-chain neutral and achiral
DNA Origami
our basic unit has been the DNA double helix with a diameter of ~2nm. 2D DX arrays should in principle have a repeat perpendicular to the helix axis of about 4-5nm but we usually find the repeat around 6nm- so our structures are designable within +-1nm. if we loosen up our design criteria a bit, to about 6nm, it's possible to design larger structures- this technique is DNA origami Image: A. design B- trajectory of scaffold strand shown as zigzag line C- holding scaffold strands together with staple strands D- helical details of structure origami is a great way to increase the addressable area (or volume) of a DNA construct
Extending bricks to 3D
rather than using strands of about one turn, they shorten each segment to 8 nucleotides, so that it represents 3/4 of a turn. This leads to an orthogonal array. 90 degree twist The idea of origami and bricks is that large constructs are entailed and the precision is high. if it's one turn, everything will be flat (same plane), if it's 3/4, you go into the 3D because helix points out.
1D Ligated DX arrays
you start with a DX tile and a triangle (without the triangle you just see a straight line) the triangle allows you to see how they point out of the arrays and since there is a 1/2 turn offset, one will be up and one will be down) A. an extra DX tile is added as a control experiment- you modify your own system and expect the same modification- so you add extra DX and get double spacing B. another control experiment to make sure that you are getting the zig zag not as a coincidence but with logic that can get you a straight product as well.
Autonomous Walker Part 2
Actual steps of the cascade worker
Striped Arrays
B has a hairpin so this allows you to visualize the stripes. Each tile is 16 nm so you see stripes every 32 nm in a and every four tiles in b.
Schematics of patterns made by capturing different molecules
Can capture any one depending on the state. Triangles point towards or away from notch. Diamond or bar- co-linear with cassettes
Calculation Restriction
Figure 12-10 shows the restriction of the reporter strand for the calculation in Figure 12-9.
PX-JX2 device in cassette
this device was converted to a cassette that could be fitted into the slot by addition of extra helix. cassette consists of device with another domain added to it on one end. The PX device is formed with crossovers between strands of the same polarity, but the cssette has been added by fusing strands of the opposite polarity. Add hairpin marker- appears as circular magneta helix with yellow base pairs viewed down its axis. (in front of cassette device in panel a) Depending on the state of the device- the hairpin willbe pointing to the back or forward
How can you modify surface of DX arrays?
- restrict - anneal/ ligate - DNAzyme (auto digestion)- Copper yields auto digestion of the hairpin on the B' as seen in the previous image You can add a complement to B' tile so you can anneal or ligate (coordinate bonds between hairpin and single strands of B_ so if you have 64 nm spacing and you add hairpin, the spacing will decrease to 32nm
DNA Walker
- Sidewalk is a TX molecule - Walker is two domains of DNA connected by flesxible link - Has front and back foot - Was held down to sidewalk (two set strands) - If you release one using a toehold, you could flop around, - if you add another set strand, that would bind front foot - can release back leg and repeat the process - similar to inchworm So, they change their position relative to an external sidewalk as a result of a structural alteration. They can move from one place to another so the sidewalk is the key element of the walker. it would just flail around if two of its legs were not able to attach to a sidewalk and then ot change their positions of attachment. KEY THAT WALKER NEVER CEASES TO BE ATTACHED TO SIDEWALK
Tensegrity in Art and DNA Origami
- combination of tension and integrity - Large aluminum rods held in configuration by tight strands - In DNA strings are single strands of DNA - 13 helical bundles Tensegrity Triangkle: Symmetric traingle with sticky ends 21 bases (2 turns per edge)- stress system by reducing numbers of bases per turn- can make 3 and 4 turn triangles 3 turn; 2 turn- 7 bases, will have 31 bases per edge or 32 since 10.5 * 3= 31.5 if you have 32 then you would use 18 bases between the junctions 4 turn- 42 bases per edge (10.5 *4=42) 7,17,28 bases between junctions
Origami Box
- has 6 faces that are almost all the same size - one single origami - the box has a lid and the lid can be locked- there are two sites that lock it. blue strand- short paired with long and yellow strand- the same but with a fret pair Fret signals when box is closed! - if you take the key and pair it with long strand-toehold- and the box will open Six segments of the M13 viral strand are color-coded, and these colors correspond to the faces in panel b dimension 36 x 36 x 42 nm - enough to hold a macromolecule and thus is implicated for potential utility in macromolecular drug delivery. can fold lid off the box leaving one edge intact- kind of like a jack in the box. you can open the lid selectively because two locks have been installed. so locks that pair with each other but can be removed using basic toe hold technique
Why poor resolution?
- nature of freezing process for stick-like motifs - inherent floppiness of DNA - need of the components of a lattice to find their own free-energy minimum
Origami to make patterns
- no guarantees that every staple strand is present in every origami molecule. - The characterization of origami constructions is usually limited to "cherry-picking" in AFM or TEM experiments.
Multiple Molecules Per Asymmetric Unit
Ability to control unit cell contents by means of sticky ends has enabled construction of crystals with more than a single crystallographic repeat. The triangles in the picture do not associate with themselves but they associate with each other The ability to control the contents of the unit cell gives us the ability to control some of the properties of the crystals The triangles themselves are two turn symmetric triangles
Wang Tiles
Algorithmic Assembly- idea is to use DNA motifs (tiles) as logic gates that can be used to produce patterns with fewer tiles than are absolutely necessary upper portion: 23 small tiles with differently colored edges bottom portion: shows how they can be assembled if one requires every tile to assemble by having similarly colored The pathway meets another vertical pathway desce edges bind together (red with red, green to green, etc.) assembly shown which is unique if tiles are not allowed to rotate, demonstrates addition (5 +9 = 14) Starting from the upper left corner, green blue diagonal assembles, while white vertical path starts at fifth (5) tile in top row. Where they intersect at bottom, the blue/red/orange/white tile is inserted and the pathway changes from a diagonal to a horizontal pathway as a consequence of orange-orange interactions. the pathway meets another vertical pathway descending from the ninth (9) tile in the top row. Where they intersect, orange/red/purple//white tile is inserted so the direction of the pathway is changed again Purple-gray diagonal intersects top row at fourteenth (14) tile At bottom- there is an 84 tile array but only requires 23 unique tiles. Saving of a factor of 2.6 (3.6*23 = 84) If this assembly was made using this system rather than individual tiles, this arrangement
Error Free Binding Protocol
All capture molecules are competing with molecules who's sticky ends are wrong and two molecules whose sticky ends are half right. have to do something so it works and you don't get mistakes- if you add molecules step-wise (sequentially) you'll get correct and half correct to bind and incorrect will not. then you heat and add the next, so the correct will displace half correct. go through 4x of them then you will have the correct one. Algorithmic assembly treats tiles as logic gates- allows you to build things you wouldn't be typically able to build.
Origami Predecessor
An immediate predecessor to DNA origami9.5 is shown in Figure 9-5. This is an octahedron that largely folds on itself, using the concept of PX-cohesion (see Figure 3-12).9.6 However, only seven of the 12 edges consist of PX DNA. The other five edges are DX (DAE) motifs, and they are formed by the five cyclic blue strands visible in the image.
More Arrays
B and D have hairpins which is indicated by the dot, and the hairpin on the B' contains sequence that can be cut by a restriction enzyme. when you restrict, you will have a space that is double as wide so the original 32nm spacing will be 64nm. you can only see double strands on AFM, you only have single strands on B tile, so they won't be visualized using AFM
Z-DNA
B-Z transition of DNA in image Z-DNA is a left handed DNA. so the yellow stuff can become Z DNA if it's in the right conditions and there are 20 nucleotide pairs in this case but otherwise it's BDNA like the rest. It's bonded to DX motif and a pair of DAO's When we turn it into ZDNA will give a major change. the BDNA gives us 36 degrees whereas ZDNA is about 30 degrees. It corresponds to a change in about 3.5 turns- the right piece of red and blue changes from below to top because of the change in half turns.
Bowtie Junction
Bowtie junctions are variants of the conventional Holliday junction in which the crossover strands contain 5', 5' and 3', 3' linkages. The remarkable thing about them is that they form junctions in which the non-crossover strands are closer to parallel than to antiparallel. In the drawing below, (a) shows the angle convention, along with slightly distorted antiparallel and parallel ideal junctions. (b) shows the differences between normal and Bowtie junctions. Note that the Bowtie junction has 5', 5' and 3', 3' linkages in two of its strands. (c) below shows what these molecules would look like if they were ideally antiparallel or parallel. In Mg(+2)- containing solutions, the normal junction looks like the antiparallel junction on the upper left, and the Bowtie junction looks like the parallel junction on the lower right. The key feature is that both prefer a chain-direction-reversing structure for the crossover strands.
PX-JX2 device
Break the red strand, put in the green strand, break the blue strand, put in another green strand. The horizontal lines are toe holds so you can toss in the complement and it will pair with the green strands and yank the green strands out. The black dots are biotin groups which can be easily removed with magnetic strapdavadin beads which bind to it. So, you can take solution and shake with beads and then put the magnet in and beads will come to the bottom and then you decant the solution and leave the magnetic beads behind so you get a naked frame. you can make different control elements to make different versions of the naked frame (anything that is different where the green strands binds is the control emlement) so you can have a bunch of these devices ina pot and control one without controling the others you have 8 states for your 3 devices (2^3=8) When yellow strand is tossed in, JX2 is flipped by a half turn
First DNA Nanomechanical Device
DNA circle- has a branch junction at the top and this junction is a semi mobile junction (there are four positions/ technically five where the bases are related by a 2 fold axis of symmetry. If you change the effective stress, you get the circle to move and stress was changed by adding an intercalator.
DX Arrays
DAO and DAE can be used for DX arrays. DAE-E has a circular strand. the E and O on DAO is the spacing in between the tiles and this can be analyzed with an AFM
Capture System
DNA origami simplified combination of devices and structures immensely. Rather than combining large number of tiles to create area of adequate extent to demonstrate the motion of a nanomechanical device by AFM, it is only necessary to build a single origami superstructure on who's surface the action of the device needs to be shown. *early example of utilizing DNA origami tiles as support for molecular motion entailed insertion of 2 different PX-JX2 cassettes into a single origami tile to create a capture system for a variety of shapes Array= DNA origami tile acting as superstructure for the system. PX-JX2 device anchored into origami tile by domain DNA duplex, drawn as green box with sticky ends to the left and right of the box. There are two such anchors, one on left and one on right. Sticky ends are labeled A and B and C and D. When it's JX2 the C and D get flipped so you can capture something different.
Paukstelis DNA Structure
DNA that's not simply watson-crick robust motif discovered in a single crystal structure this motif contains three conventional nucleotide pairs and three parallel pairs consisting of one AG pair and two GG pairs. When looking at it in stereo, we see that the double helical arrangement is quite distorted. The Watson-Crick portion of this motif can be extended by 10 nucleotide pairs, yet the space group remains the same. Although the resolution of the crystal decreases, the cavity is expanded by this expansion. Notes: tried to crystallize this long RNA molecule and long DNA molecule and a 13mer in the structure. formed crystals very readily the x means you're switching from one strand to another G6 is paired anti-parallel with A7 so in the box, everything except for in c4g9 is not Watson crick
Origami Patterns
Decoration of an origami surface to produce patterns. By adding transverse hairpins (see panel d in Figure 9-1), images are created. This is arguably the greatest importance of DNA origami, the addressability of ~8000 nm2 in about 100-200 positions. The most important thing about DNA origami is not that DNA strands can be folded into smiley faces or the other arrangements shown in Figure 9-2. Rather, origami gives a way to address something approaching 10 000 nm2 at about 200 or more loci. The spelling of "DNA" and the triple hexagonal pattern would both be extremely difficult to do with groups of individual tiles.
Standalone Nanomechanical Devices
Devices that are not combined with other DNA constructs
Solving Hamiltonian path with DNA
Each city has a 10 nucleotide first name and a 10 nucleotide last name Each of the yellow citcles on the 5' end of the 20-mer representing a city represents a phosphate. The phosphate allows strands to be ligated to other strands. Route is shown on lower right. Complement to the last name of the origin city fused to the complement of the first name of the destination city. ROUTES ARE UNPHOSPHORYLATED.
Unstressed Origami
Everything more or less relaxed - they are changing the separations between the circles so that the smaller ones can be nested inside larger ones (everything isn't held together perfectly) - Circle and supercircle - all 2D - Series of circles --> spherical arrangement (or ellipsoid) - All simple and straightforward to do with origami - Nanoscale flask (skinny up top and then widens out. looks like a vase. )
Intercalation
Has to do with flat armatic and greasy drugs. They have a bunch of bases (drawn esssentially in ladder form on the diagonal) and the bases are 3.4 A apart, 6 bonds in the repeat (1.5A) each 9A to play with but not exactly true because they're bent 109 degrees or 104 degrees- about 7A distance between backbone (so that's about double )
How Walker Walks
Held on position by strand A1 and A2 as well as orienting strand A4, fuel (unset) strands are then added to remove A1 and A4 so it's only attached by A2. Then new positioning strand A3 is added to the walker so it's now attached by strands A2 and A3 and has advanced 120 degrees. (1/2 step)
Bowtie Junctions cont
How do we know that these molecules are parallel, rather than antiparallel? Preliminary evidence was obtained from gel mobility studies, but the more interesting demonstration was provided by AFM. Neither normal nor Bowtie junctions are ideally parallel or antiparallel. Rather, they differ from these structures by about 60 - 70 degrees. If the diagram below shows that a V-shaped array made from I, III, and IV will have an acute angle if parallel, and an obtuse angle if antiparallel. Likewise, a V-shaped array made from II, III and IV will have an obtuse angle if parallel and an acute angle if antiparallel.
3D Origami Containing Stress
If you want to make 6 helical bundle --> crossovers 7 nuc pairs apart or 14 nuc pairs apart 2/3 or 4/3 of a turn (relaxed) Less than 7bp have torque in one direction - opposite direction if more than 7bp Can take relaxed stuff with stressed stuff on either side --> bend it into an ark (torquing it) Twisting a lath of DNA - can twist in left or right handed fashion Can stress to bend - depending on how much stress you put on red region you can bend (all the way to 180 degrees into a U shape) you can distort the DNA origami and it has to do with branch migration and crossovers of molecule twisting has to do with how many times backbone goes around helix axis. ride is distortion of helix axis. ride is more complex. you can stress it to get it to bend and depending on how well you design system, you can get it to bend from 0 to 180 degrees. can form a circle or a spiral etc. can make something that looks like a gear Twist: how many times backbone goes around helix axis Ride: more complex a way in which the DNA will respond the stress --> bend (distortion of the helix axis - bending of the helix axis) Ex. Midline of belt is helix axis - don't have to close into a simple circle, can turn into negative supercoil by twisting belt before looping, the helix axis is almost undistorted which there is a twist. Can take the same thing and radically change shape (eliminating twist) but the change is the ride by making concentric loops. Why you can separate topo-isomers on a gel (radius changes) 7 nuc pairs b/w connections --> lattice like structure Fewer nuc pairs --> will twist in one direction More --> will twist in the other direction Can distort origamis* all has to do with crossovers and branch points
RNA Square
In the figure, four molecules with a previously seen bend are combined into a square using sticky ends.
PX to JX2 shift
It should be clear that in the PX state the indicator helix is pointing in towards the marker tile, while it is pointing away from the marker tile in the JX2 state. So PX state is seriers fo cassettes- every other column with hairpins pointing down the marker JX2 state is change state of device and cassette so you flip by 1/2 turn and will flip up this change is what's observed in the AFM.
RNA Trefoil knot
Knot was prepared similarly to how DNA Knot was prepared. Single strand prepared containing two potentially paired domains a single turn long. On the left: ligation takes place in presence of a linker strand too long to accommodate two turns- product is circle On the right: linker is short enough so that knot is formed It was used to search foa topoisomerase activity that works on RNA. It was found indeed that e.coli DNA topoisomerase 3 (not 1) has this activity.
Protein independent walker (Inchworm)
Moves like inchworm Its sidewalk is simple- ends of a TX molecule. The TX molecule is shown as 3 doubly connected rectangles at the bottom of each of the six panels in navy. The walker consists of 2 double helical segments of DNA drawn in yellow that are connected by a flexible linker. There is a foot 1 and a foot 2 which can rest on positions called foothold A and foothold B and C (unoccupied in first panel) The attachment of foot 1 to foothold A is achieved by set1A and same for B - set 2B. Both of these strands contain toeholds(the horizontal extensions) Part B you get unset/ fuel strand added to the system. C- set 2B has been removed from the system. small blue circle represents biotin group that can be used to remove duplex of strands set 2B and unset 2B from system so you release foot 2 from sidewalk but foot 1 remains intact. new strand set 2C added to system so now the walker has extended itself like an inchworm. in e, set 1A has been removed by its unset strand (analogous to state c) but in this state, foot 2 is on sidewalk and foot 1 is dangling. The red bottoms of the feet are psoralen groups- used to crosslink the walker to sidewalk in various positions - demonstrate walker has taken place. ; another walker was made and demonstrated by FRET data rartehr than crosslinking CLOCKED DEVICE- does not function without iintervention of some sort of operator.
Tensegrity Triangle
Note the three-fold symmetry of the sequence, and the rhombohedral nature of the crystals. These crystals are macroscopic, and can be seen with the naked eye. Over and under nature of structure directions of the three helices are linearly independent and so they span a 3-space there are covalent crossovers btwn the three helices but they're not readily aparant. so anyways, these are triangles molecules containing 63 nucleotide pairsand they migrate accordingly ona non-denaturing gel. this is a rod-like structure.
Molecular Assembly Line
One of the holy grails of nanotechnology has been the molecular assembler to build new molecular species from the bottom up. The programmable assembler is the same PX-JX2 cassette but this time it's connected by 2 helices (double cohesion rather than by single sticky end pair. Three of them are lined up and a conveyor and product has been combined into a walker that somersaults along pathway and grows as items are added to it. walker is tensegrity triangle. In the first part where it's all JX2, the casettes are in the off state because as we remember, JX2 points away from the marker tile and in this case the walker. . It takes half steps after it's turned on and the final step is the release of the completed complex. the three cassettes have to be sunk into the origami so there's a domain of these cassettes and that are in the plane of the origami and those are made of strands that have to be good. when we talk about origami we usually talk about strands where we take what we can get from the supplier and toss these strands together along with the scaffold into a pot, cook em up and cool em down, that's true for the strands that only talk to the scaffold but the strands that are going to talk to the cassette and the strands that comprise the cassette are both purified. they have to be just the 36mers and none of the 35, 34, etc. *Based on proximity!!!
Thermodynamics of Walker
Panel c shows how proximity of the C1-bearing arm leads to its attachment to the walker. First there is cohesion between the unpaired segment attached to C1 and an unpaired part of H1. Following that, the second step entails branch migration so that more base pairs are formed between C1 and H1 than between C1 and the strand attached to the cargo station. **Note that there are more nucleotide pairs formed at each of the successive steps of panel c; this is the thermodynamic driving force for the cargo transfer. Cargo is attached to purple part. The olive part is what binds to the red part. and then the H1 will displace the duplex. So now you have attached the cargo to this H1. favored because more base pairs are formed than in the second state. H1 makes more base pairs with that magenta strand than were made beforehand so that's the driving force.
DNA Bricks in 2D
Start with a single stranded tile (use a bunch of U shapes for a cylinder) Built a molecular canvas Series of bricks in canvas - there are two kinds of bricks a whole and a half brick. The whole is a U shaped guy, the half is a half of that (helps smooth things out) Take the corresponding bricks and throw away (to make eagle head) add half bricks to smooth out areas in order to have a nice flat line Once the canvas was put together - can choose to leave out or add half bricks any of the bricks on the canvas Cook it up --> cool it down (gently & slowly) Can make anything - a matter of throwing away tiles from the 2D array Haven't distributed the software widely
PX-JX2 Topoisomers
Switching rotary device of the bottoms but not the tops. PX: two molecules are talking to each other JX: juxtaposition of backbones; isomer of PX. Don;t have a turn and a half but simply one turn. B and C are on the same side rather than A and C in the PX. The tops in JX are the same but the bottoms are flipped by a half turn
Casettes
TX array can be connected 1-3 to its neighborus- generating gap that provivides little space for accommodating attachments. this space provides attachment point where nanomechanical device can be placed Doesn't always have to be TX arrays, doesnt' really matter. But it's that thing with the device.
TX
TX has three domains (DX) so it will have a space that is important in relation to inserting devices in the array, int he array that you don't have in the DX. The red lines indicate C that was inserted perpendicular to the others- C' is rotated D is connecting in between the B's and is a duplex. So there are three TX tiles and one duplex.
Key to solving the problem
The key point here is that T4 DNA ligase only ligates two molecules separated by a nick, not just any two molecules in the reaction vessel. Thus, the ROUTES ACT AS CATALYSTS TO PROMOTE LIGATION of any two 20-mers representing successive cities for which a route exists, but a pair of cities for which a route does not exist will not be ligated together. The notion is that all pairs for which routes exist will be joined, but the ligase will not stop there. Every possible route of whatever reasonable length will be formed by the ligation reaction. The correct answer will be 140 nucleotide long. This can be separated from other strands by running a gel. We also know the correct sequence had to begin with Quito and end with Shanghai (part of the problem) Strands not starting with Quioto and ending at Shanghai would fail to be amplified by this procedure. The presence of the other 5 cities had to be determined individually by binding the candidate strands to beads that contained complements to those cities. Pull out beads and everything that wasn't pulled out wasn't that city. so he did that fives times.
2D Origami Lattice
The most successful approach to building 2D origami lattices has been to orient the helices in the directions of the unit cell. Figure 9-6a shows this strategy, with helices pointed perpendicular to each other. Figure 9-6b shows a zoom of a portion of a 3 micron × 5 micron origami lattice. It should be noted that this was also the successful strategy employed in Chapter 7 to make 3D lattices: propagation of the lattice in linearly independent directions that are parallel to helix axes. Combined a number of motifs that had the same pattern as an origami where all helix axis were parallel to each other --> if all axis went the same way you had a problem - should design an origami that is cross shaped --> half helix axis would be vertical other half would be horizontal Basic signature is that you have a double thick layer (equal sign on the inside) - about 2 or 3 microns Want to have helix axis pointing in both directions --> same in 3 cases i.e. tensegrity triangle can form crystals in 3D
Ethidium
flat aromatic molecule which is similar to the bases that can intercolate in between bases at 7A. Therei s space to put a greasy drug into the space in between a given bsse pair- it separates them about 7A but the DNA unwind. Let's say there were 36 degrees in B DNA double helix, 6 degrees unwinds the DNA helix about 30 degrees which relaxes the DNA (not extruding) so unextruded will suck the 8 base pairs into the circle like the image on the right.
Analytical Devices
They measure the energy of binding a given protein to a given sequence of DNA Here you have 2 TX's connected by a shaft and they measure protein IHF (integration host factor: recombination factor- a viral integration into DNA) which bends DNA upon binding. To get from Da to Db, you have to rip the sticky ends apart and the IHF has to be strong enough to do that. You can change sequence and length of sticky end pair. There is also a fret pair (donor and acceptor) The IHF- driven device is not entirely general, it depends on the position ont he DNA to which the protein binds. a more general device based on a scissors like motion has been developed to analyze the binding of MutS protein. MutS is a DNA-repair protein that recognizes mis-paired or unpaired sites in DNA. When it binds to such a site, it bends the DNA. It's large so it has to distort the DNA hence the scissors developing. So this device is a pair of DX domains connected by a single crossover site in the middle which acts as the fulcrum of the scissors. The blue section represents the DNA duplex to which the MutS will bind and the magneta portion is the lesion that MutS recognizes. Going from d to c is a 90 degree rotation about the vertical. e- mutS binds to its binding domain and closes the scissors. just like IHF, ability of mutS to close blades and alter FRET signal s function of how strongly it binds to binding site. The different possible lesions that are recognizzed by mutS are characterized by different binding energoies and these are in turn reflected in the strength of the sticky end that MutS is able to separate.
Diversity of Products
This is a programmable system. There are 8 different pathways through the construction cycle, depending on how the cargo stations are programmed. the eight possible products are shown at the right including no addition of cargo, three pairs of cargo, etc. There are 3 differentn control strands so it's not just a matter of having every machine all do the same thing at the same time. IT's the programabiltiy and orthogonality of those states by uusing different sequences in control regions that can supply control. If you only had BZ device, you could either add them or all, or not add any. This gives a variety.
3D Structures
This is done by using a model-independent method known as isomorphous replacement, rather than by using a method known as molecular replacement, which requires a model. Crystals were based on tensegrity triangles. The tensegrity triangle spans a 3-space so it's good for a 3 crystal. The two characteristics of the failure crystals were long sticky ends and 2D motif that was tricked into makeing 3D arrays. ex. combo of 2 TX Molecules joined by short length of duplex that had non-half-integral twist between junctions sticky ends that provided best resolution were only 2 nucleotides long.
I-Motif
This is the structure formed by hemi-protonated oligo-dC. The requirement of a relatively low pH to protonate the C nucleosides makes it a useful tool for estimating the pH within the cell. Device that contains olig-dC in a single strand that can fold to produce the I-motif Device contains FRET pair of dyes whose average separation is dependent on the formation of the I-motif When unfolded as on the left of the iamge, they do not transfer energy but when folded as on the right, they produce energy transfer whose extent can be measured- device is sensitive over pH range of 2 units
Toe Hold
Toe hold is the hanging strand that can be displaced. So in the tweezers diagram, F is the toehold. strand F bar is the complement to strand F and it binds to F which then branch migrates (isothermal change) Single stranded branch migration- there ar emore base pairs in the FF' duplex than in the F molecule being paired with the rest of the device so it is thermodynamically favorable to make that DNA duplex that leaves the system. If you have n steps, after n^2 events, you will have bound F' to F and will have duplex as a waste. This duplex won't come apart because it's a duplex (irreversible isothermal process) and it's favorable. There were some dimers after one cycle The whole system works because of strand Fˉ, whose sequence consists of complements to the red portion, to the green portion, and to the blue portion of strand F. When strand Fˉ is added to the solution, its toehold binds to the toehold. Then, through single-stranded branch migration, it works its way through the green and blue sections of F, displacing it from the rest of the tweezers. A duplex of F and Fˉ is formed, and this is waste for the system. The open state is restored. It is key to realize that the toehold-mediated strand removal is isothermal, requiring nothing more than the addition of the Fˉ strand (often called the "fuel" strand) to reset the system. Virtually all enzyme-free nanomechanical device systems are based on toehold-mediated strand removal. Although we described it earlier, it should be clear that the G4 device of Figure 8-4 was based on this principle: that's how strand β removed strand α.
AFM of the PX JX2 device (two state device)
Trapezoidal half hexagons- single triangles with DX on one side is connected to device. Top row is PX state (strands for PX state are green) then toss in complements for green strand then add yellow strand and it switches from state where triangles are parallel to where one is pointing up and the next is pointing down. You can switch between the two.
PX-JX2 device in cassette cont
What do we have to do to make 2D array design? These are 8 different DX molecules, the brown and red one and green and blue, purple, and pink, etc. in order to be able to put together enough DNA so you can see the hairpin flipping from on eside to the other in the AFM, we didn't know how big to make it. could have gotten away with maybe only 6DX molecules. have to make sure that things that will talk to each other needs to be purified. Cassette inserted vertically into array is red. One tile away another vertical tile is black and labeled M- marker tile. This is inserted so that up-down directionality can be established so you know which way the indicator helix is pointing.
RNA Origami
further step- produced a single-stranded RNA structure that folds and combines with the other molecules to produce hexagonal lattice.
Walker Structure
hands: H1, H2, H3 at one end for grabbing cargoes. feet: F1, F2, F3 for walking on other ends of domains. Foot F4 is used to orient the walker towards the cargo loading stations.
3 State DNA Based Nanomechanical Device
Wound up with not only three states but also three transitions First and second state rotate the ends of the device Can rip out set strands (from either state) and can replace them with purple strands (they do something very similar to the closed tweezers) will bring these strands together --> contract the two so it will be shorter Will be the same as in the JX state only shorter (1.rotation,2.translation, 3.screw rotation (two fold screw rotation: rotate a half turn and negate that translation)) Screw rotation very similar to fat molecule forming alpha helix- rotate 180 degrees then translate
XOR Tiles
a. TX tile that is used to represent the units red reporter strand has been drawn with athicker line to emphasize importance b. illustrates tiles used in the assembly. two light blue input tiles, two green initiator tiles, and four red XOR tiles. bottom domains of the XOR tiles represent their recognition properties: left to right, (0,0)-->0 (0,1)-->1, (1,0)-->1, (1,1)-->0 these values are coded in their sticky ends which are drawn as different shapes. Same sticky ends are used for 0 in both cases and for 1 and then when youre looking at xi same trend. youre looking at yi-1 and xi
RNA
alternative to DNA. RNA double helix is A form rather than B-form. some of the unusual motifs available to DNA are also available to RNA but rarely does RNA nanotechnology use same sticky ended cohesive approach as DNA nanotechnology does.
Rhombohedron
another way to think about it is that it forms a rhombohedron, this is a cube like structure inw hich one of the body diagonals has been stretched or shrunk, leaving polyhedron with only a single three fold axis, rather than four of them. The center of a triangle drawn in red flanking rear vertex= connected by sticky ends to each of the yellow triangles that flank it and whose centers lie in a place closer to you. yellow triangles are connected to the three green tirnagles even closer to you. there is a second red triangle above the first one
PNA-DNA Duplex
double helix that's not the same as the DNA-DNA double helix. Only the yellow and green strands of the A tile consist of PNA the shaded nucleotides of the B tile can be removed or extended in the experiment. vary lengths of those segments- different effective helicities- control
Origami 3D Lattice
extension of origami to 3D was based on 6HB notion of 120 degree angle resulting from using 7nucleotide separations to orient collection of helices in 6HB
Bar code system
first system to combine a long strand with helper strands bar code has strand that spans five tiles, and the tiles are selected by the sequence of the bar code. *Different long strands can select different bar codes, depending upon whether the tile contains a pattern marker or not. You make a 1D DX array but you can put together a single long strand (red strand) and that can be coded to bind either the unit with the feature or the unit without a feature On - off - on - on - off (b) Simple form of coding
XOR Calculation
first tile X1=1 and two initiator tiles C1 and C2 assemble, forming a slot for the first answer tile (Y1). The value on C1 is set to 0 so the Y1 tile is a 1 tile. This value is reported on its upper right sticky end. When combined with x2=0, the y2 is also a 1 tile. Combining this with x3=1, binds y3, a 0 tile. The arrangement is read out through the reporter strands shown, for the region flanking the initiators, in the lower right panel of the figure. The reporter strands are ligated together to produce long single stradn connecting input to output. Each of the tiles contains restriction site on reporter strand for the two possibilities 1 and 0.
How does DNA Origami work?
take single stranded M13 virus (scaffold) and combine with ~250 staple strands to get it to fold into a series of shapes containing parallel helix axes. These shapes are based on the extended version of the DAO motif. The basic idea is that short strands can fold the long strand into the target shape. -Very different scale (around 7200 nuc) -Cyclic strand of DNA around 7200 nucleotides long - often in 13 single stranded form -Sequence is determined by m13 *a virus* (you're stuck with the m13 sequence but it seems to work) - everything you do must conform to the sequence -Take scaffold strand --> folds m13 into Christmas tree shape --> scaffold strand - black staple strands - colored --> everything will pair together -As long as your strand is only talking to the scaffold you don't have to purify it -Looking at the construct on the AFM -Scaffold strand that you want to design into overall shape - find staple strand that will hold it into the shape (part e) - Do not need to purify the 200 staple strands
Non-Robust Sequence Dependent Tweezers
these tweezers open and close. the red ticks are a fret pair. in order to close, blue is complimentary to the blue and green is complimentary to green and the red hangs out. you get closed formation and the fret pair will come together so you get closing of the tweezers. in order to cycle, throw in the part that will bind with the red part- you get branch migration and then the duplex DNA strand is the waste. almost every sequence dependent device works. But what makes it not robust is that the molecule seems to come apart and resassociate so different strands will come back together and you get dimers, multimers and you can trace strands going from one to next. The red part is the toe hold and has to be long enough to hold on. It jiggles one step forward, one step back, and if you have 20bp, it will take ~400 steps. not robust- it can dissociate. and form dimers, etc. one step forward, one step back, it jiggles. if you have a 20 of steps, after about 400 steps you get to the end. n^2 this is the basis for sequence dependent devices How do we know this works? The little ball and triangle represent a dye and when they're together, the system goes dark but when you separate them as they are when the tweezer is open, it lights up!
PNA DNA AB Tile ??
this is what happens when either DNA or PNA i sused with variations of those shaded nucleotides. B0 corresponds to 10.5 fold helix, B1 to 11.5 If helicity is not approximately correct, 2D AB* array won't form B0 tile that DNA forms a nice 2D array but PNA array is nonexistent with this B tile. As the number of nucleotides in the B tile is increased, the DNA 2D AB* array decreases in quality. As B tile increased in size, PNA array starts to show 2D features.
Coloring Crystals 1
triplex molecules containing dyes have been added.. Panel A- crystal with no added TFO, panel B shows crystal binding a TFO with Cy5 (turquoiise) Panel C shows crystal with fluorescein, yellow dye, attached to triangle Panel D shows TFO binding to same crystal, yielding a green triangle Cy3, cy5, tfo, central strand has a color
G tetrads
triplexes are not only unusual multi-stranded DNA structures. G4 DNA tetrad is intriguing system to be used in conjuction with Watson Crick duplexes. Formation of G4 tetrads can be used to synapse two different DNA helices. So this is a new way to join pairs of double helices just as PX-DNA s a way to do that.
Autonomous DNAzyme Based Devices
until now, we added a set strand of one flavor or another and would then choose what happened Here you take a DNAzyme which is the motive force for this device this device is autonomous which means it does not need human intervention Enzyme is a ribonuclease and it cuts RNA not DNA So you throw RNA into pot, it gets cut, you have 2 short pieces that are so short do they do not stay bound to their compliment and so they dissociate and you get tweezers. another substrate pieces comes in, and you return to the original state- cycle. so the substrate is the fuel- it cuts and squeezes whil e it cuts. There is a brake- green strand is made of DNA and it sits there and it has a toehold so you throw in a compliment to the green strand and it will form a duplex.
RNA in vivo
using RNA as a basis of nano technological constructs is that the products can, in principle, be produced within a living cell, by process of transcription. If transcription is under control of a series of promoters and various transcription factors, then the structures can be generated only under certain conditions. ex. produced a nanostructure that binds hydrogen-producing proteins in close proximity to each other Figure: RNA nanostructure that binds proteins that produce hydrogen- thereby organizing them.
Go back to chapter 7- 3D-DX triangles
valuable in 2D but not produced 3D crystals. It's based ont he Mao's group's tensegrity triangle, except it contains DX motifs rather than individual double helices. There is extensive curling in all three panels but it's fairly flat. The parallelogram arrays were not very robust when edges are made long.
G-Tetrads cont.
variation on this theme. forms a 3-arm junction with G4 at branch point. G4 can be formed from both parallel strands and a mixture of antiparallel strands. depending on the tosrion angle about the glycosyl bonds.
RNA 2D Lattices
variety of RNA constructs based on a combination of Watson-Crick base-pairing and other motifs mined from the structures on naturally occurring RNA molecules. Structures include loop-loop interactions combined with sticky ends as shown in figure number of these molecules can be combined to form 2D lattices
Not Just Vanilla DNA
work that is going on with other DNA structures, work that entails non-DNA backbones, and other species organized by DNA nanoconstructs