Introduction to DNA Origami Plenty of Room at the Bottom: a talk by Richard Feynman (1959) Offered $1K to the first person to write something that could be read by an electron microscope Offered “$1K to the first guy who makes an operating electric motor---a rotating electric motor which can be controlled from the outside and, not counting the lead-in wires, is only 1/64 inch cube.” Engines of creation by k eric Drexler Nanosystems 1992 by drexler Ned Seeman at NYU made geometric structures out of DNA (80s) Paul Rothemund at Caltech (2006)

Synthesis of DNA origami Rothemund P W K 2006 Folding DNA to create nanoscale shapes and patterns Nature 440 297–302

What DNA is? + Phosphate 2-deoxyribose Base (adenine) DNA stands for Deoxyribonucleic acid Contains the genetic instructions of living organisms Polymer of nucleotides, with backbones of sugars and phosphate groups joined by ester bonds. A base is attached to each sugar. The sequence of bases encodes information. http://upload.wikimedia.org/wikipedia/commons/4/43/Deoxyribose.png http://www.thestandard.org.nz/wp-content/uploads/2009/01/phosphate.gif http://med.mui.ac.ir/slide/genetic/dna_molecule.gif http://upload.wikimedia.org/wikipedia/commons/c/cf/Adenine_chemical_structure.png

DNA Structure

Watson–Crick base pairing The DNA double helix is stabilized by hydrogen bonds between the bases . The four bases are adenine (A), cytosine (C), guanine (G) and thymine (T). Each type of base on one strand forms a bond with just one type of base on the other strand. This is called complementary base pairing. A bonds only with T, and C bonds only with G. Hydrogen bonds are weak, they can be broken and rejoined relatively easily. The two strands of DNA in a double helix can pulled apart by a mechanical force or high temperature. http://www.bio.miami.edu/~cmallery/150/gene/c16x6base-pairs.jpg

Branched DNA DNA is normally a linear molecule, in that its axis is unbranched. DNA molecules containing junctions can also be made using individual DNA strands which are complementary to each other in the correct pattern. Due to Watson-Crick base pairing, only complementary portions of the strands will attach to each other. One example is the "double-crossover“. Two DNA duplexes lie next to each other, and share two junction points where strands cross from one duplex into the other. The junction points are now constrained to a single orientation. Suitable as a structural building block for larger DNA complexes http://upload.wikimedia.org/wikipedia/commons/9/92/Holliday_junction_coloured.png http://upload.wikimedia.org/wikipedia/commons/f/f4/Holliday_Junction.png http://www.dna.caltech.edu/Images/DAO-WCr.gif http://upload.wikimedia.org/wikipedia/commons/3/3d/Mao-DX-schematic-2.jpg

Design of complex DNA structures The first step is to build a geometric model of a DNA structure that will approximate the desired shape. The shape is filled from top to bottom by an even number of parallel double helices, idealized as cylinders. The helices are cut to fit the shape in sequential pairs and are constrained to be an integer number of turns in length. To hold the helices together, a periodic array of crossovers is incorporated. The resulting model approximates the shape within one turn (3.6 nm) in the x-direction and roughly two helical widths (4 nm) in the y-direction Rothemund P W K 2006 Folding DNA to create nanoscale shapes and patterns Nature 440 297–302

The dirty details Step 1 (in design process) – DNA double strand folded back on itself: Natural point to make a new link Place where red (secondary / non-master) strand comes into close alignment A Hands-on Introduction to Nanoscience: www.virlab.virginia.edu/Nanoscience_class/Nanoscience_class.htm

The dirty details (cont'd) Step 2 – Cut secondary strand at this point: How to reconnect between double strands? MUST take into account direction (5' => 3') of DNA backbone Making sure that connection maintains this progression (see arrows) A Hands-on Introduction to Nanoscience: www.virlab.virginia.edu/Nanoscience_class/Nanoscience_class.htm

Oh, but I forgot something major: Can't engineer segments as continuous strings! Backbone structure (phosphate => ribose) must repeat every 1/10 turn Position of cuts and unions MUST maintain this repetition A Hands-on Introduction to Nanoscience: www.virlab.virginia.edu/Nanoscience_class/Nanoscience_class.htm

Design of complex DNA structures The second step is to fold a single long scaffold strand back and forth in a raster fill pattern so that it comprises one of the two strands in every helix. progression of the scaffold from one helix to another creates an additional set of crossovers. The fundamental constraint on a folding path is that the scaffold can form a crossover only at those locations where the DNA twist places it at a tangent point between helices. Thus for the scaffold to raster progressively from one helix to another and onto a third, the distance between successive scaffold crossovers must be an odd number of half turns. Conversely, where the raster reverses direction vertically and returns to a previously visited helix, the distance between scaffold crossovers must be an even number of half-turns. Rothemund P W K 2006 Folding DNA to create nanoscale shapes and patterns Nature 440 297–302

The dirty details (cont'd) Step 3 – Connect up what will become "staple" segment: New red = Staple segment Blue & Yellow = non-staple segments OR if merge blue/yellow at appropriate point, they could become second staple A Hands-on Introduction to Nanoscience: www.virlab.virginia.edu/Nanoscience_class/Nanoscience_class.htm

Design of complex DNA structures The geometric model and a folding path are represented as lists of DNA lengths and offsets in units of half turns. These lists, along with the DNA sequence of the actual scaffold to be used, are input to a computer program that designs a set of ‘staple strands’ that provide Watson–Crick complements for the scaffold and create the periodic crossovers. Rothemund P W K 2006 Folding DNA to create nanoscale shapes and patterns Nature 440 297–302

Design of complex DNA structures In the final step, pairs of adjacent staples are merged across nicks to yield fewer and longer staples. To strengthen a seam, an additional pattern of breaks and merges may be imposed to yield staples that cross the seam. All merge patterns create the same shape but the merge pattern dictates the type of grid underlying any pixel pattern later applied to the shape. Rothemund P W K 2006 Folding DNA to create nanoscale shapes and patterns Nature 440 297–302

Synthesis of DNA origami Rothemund combined the DNA of a common virus M13mp18 , 250 helper strands and magnesium buffer. The mixture of strands is then heated to near boiling (90 °C) and cooled back to room temperature (20 °C) over the course of about 2 hours. Fig. Rothemund P W K 2005 Design of DNA origami ICCAD’05: Int. Conf. on Computer Aided Design pp 471–8

Visualization of DNA Origami Atomic Force Microscope

But the design programming sounds REALLY complex But there is now a free, downloadable, do-it-yourself DNA Origami design program! Developed by the Dana Farber Cancer Lab & Harvard's Wyss Institute www.cadnano.org

Small Smiley Faces Are Sort of Interesting but What Else Can I Make That Is Useful Gray = long master single DNA strand Orange + blue = Short DNA "staples" locking in master strand's 3D shape Yellow = Staples with ends hanging out, attaching to purple "cargo" Plus short DNA extensions at far-left / upper-right You can build a nanorobot!!!! http://www.virlab.virginia.edu/Nanoscience_class/lecture_notes/Seeing%20at%20the%20Nanoscale.pptx A Logic-Gated Nanorobot for Targeted Transport of Molecular Payloads. SM Douglas, I Bachelet, GM Church (2012) Science. 335:831–4

Short DNA extensions serve as LOCKS to fold structure together Twisted locks work because they consist of a pair of complementary single DNA strands HOWEVER! In each of THESE two locks, ONE of the DNA strands is very specially selected So that, when unwound and disconnected, it curls into a complex shape That can fit tightly around (and thus latch onto) a specific foreign molecule THESE DNA strands were chosen to latch onto protein antigens That are released by or embedded in the cell membranes of CANCER cells Appropriate DNA recipes are cataloged in databases for specific antigens http://www.virlab.virginia.edu/Nanoscience_class/lecture_notes/Seeing%20at%20the%20Nanoscale.pptx

These antigen-binding single DNA strands are known as aptamers When the lock springs open it exposes the antigen-releasing cell to the nanobot's deadly cargo If the antigen that a lock's amptamer targets are encountered: That aptamer/DNA strand will untwist and instead latch onto the antigen and this "lock" opens If the antigens for BOTH locks are present, BOTH locks unlock, and shell springs open! It even works if antigen spacing on target cell does not match separation of the two locks: 1st lock springs, then nanobot wiggles around cell surface until 2nd lock springs You can use build cascades of these nanorobots to release “cocktails” of drugs in response to different types of cancer or to perform in-situ surgery Amir Y, Ben-Ishay E, Levner D, Ittah S, Abu-Horowitz A, Bachelet I. Universal computing by DNA origami robots in a living animal. Nature nanotechnology. 2014;9(5):353-357. doi:10.1038/nnano.2014.58.

We Could Also Start Designing Dynamic DNA Nanomachines Dynamic DNA devices and assemblies formed by shape-complementary, non–base pairing 3D components Thomas Gerling, Klaus F. Wagenbauer, Andrea M. Neuner, and Hendrik Dietz Science 27 March 2015: 347 (6229), 1446-1452. [DOI:10.1126/science.aaa5372]

Resources CadNano: http://cadnano.org/ DIY AFM: http://lego2nano.openwisdomlab.net/index.html Solve for X - Ido Bachelet - Surgical Nanorobotics: https://www.youtube.com/watch?v=aA-H0L3eEo0 BioMOD Previous Years Projects: http://openwetware.org/wiki/Biomod biomod.net