Biomod/2012/UT/Nanowranglers: Difference between revisions
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This process can continue as the track is lengthened and as long as there is fuel available for consumption. However our design incorporates elements that limit the length of the track. Almost all things observed in nature tend to follow exponential curves. This is no different in the reaction times for DNA strand displacement. We know from observation that not all walkers will complete their first steps at the same time, and some may not even complete one. This is due to the subtle differences in free energies of the displacements that our design employs. Because the drive is not very strong, we expect the walkers to decrease in number as they continue moving forward due to energy dips and mismatched base pairs. | This process can continue as the track is lengthened and as long as there is fuel available for consumption. However our design incorporates elements that limit the length of the track. Almost all things observed in nature tend to follow exponential curves. This is no different in the reaction times for DNA strand displacement. We know from observation that not all walkers will complete their first steps at the same time, and some may not even complete one. This is due to the subtle differences in free energies of the displacements that our design employs. Because the drive is not very strong, we expect the walkers to decrease in number as they continue moving forward due to energy dips and mismatched base pairs. | ||
='''Fueling the walker'''= | |||
Directional walking cannot be achieved without a source of energy, such as a fuel molecule [cite Kelly 2005]. The fuel molecules we designed transmit energy to our system via the CHA (Catalytic Hairpin Assembly) reaction [cite]. | |||
[[Image:CHAdiagram.png|thumb|right|300px|Diagram of a CHA reaction. The initiator opens the red fuel which exposes a hidden toehold binding region. The blue fuel removes the initiator from the red fuel, regenerating the catalyst.]] | |||
<span style="color:red"> BB: Andy, please copy the contents of CHA-kinetics.docx here. </span> | |||
We built a bipedal walker by hybridizing two strands of DNA that have overlapping domains on both sides. | |||
The walker utilizes a track that is composed of eight strands of DNA. Two make up the base of the track while the other six are hairpin structures (DNA that partially binds to itself) that are hybridized to the two base strands forming a long duplex with alternating hairpins along its length. These hairpins will serve as the footholds for the walker to attach itself to. The walker is driven to walk by repeated instances of the CHA reaction and a series of toehold-mediated stand displacement reactions. After the walker has taken two “steps” the second hairpin on the track will have a long duplex that a nicking enzyme will cut specifically. This will cause both pieces to dissociate from the track due to a lack in strength of binding. Because these fuels are added once to the system at the same time, and are removed from the track by a nicking enzyme, the walker is both autonomous and non-destructive to the track. | |||
<span style="color:red"> Why is the above text here??? </span> | |||
='''Acknowledgements'''= | ='''Acknowledgements'''= | ||
Revision as of 12:42, 27 October 2012
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Concept
Motion is a necessary component of life. While this does include the motion of animals and people as they move about, there is also crucial motion which takes place on a cellular level. Cells could not function with the proteins kinesin and myosin [Cite Mehta et al., 1999], which carry cargo to and from our organelles. The movements employed by these proteins are very similar to the way people walk, in the fact that they have two “feet” which step forward, one at a time [Cite Vale et al., 1996]. Because of the crucial role these proteins play in supporting life, we began to wonder if we could create our own molecular walker that could perform similar tasks. In order for this walker to be comparable to, or even better than, kinesin, it must be autonomous and run on a reusable track. In other words, it should not require an external driver and should not damage the track while taking each step. Other walkers have been created in the past, but none has yet been able to accomplish both of these tasks. There are, however, some that are at least capable of either walking autonomously [Cite Bath et al., 2005] or reusing their track [Cite Yurke et al., 2000], even though they cannot do both. Our walker is able to accomplish both tasks by means of two alterations. The use of two discrete fuel strands which can only interact with the walker system in a specific order allow us to add an excess of fuel without causing the walker to fall off the track. We also make use of a nicking enzyme which restores the track to its original form without harming it, allowing it to be reused.
These genes were made for walkin'...
Design overview
Our walker design consists of four main components: a walker, specifically a DNA molecule with two "legs" and a body capable of storing cargo; a track, a long platform supporting footholds to which the legs of the walker can attach; fuel molecules that supply energy to the walker system; and a nicking enzyme which reverts the footholds along the track to a usable state after the walker has walked across them. The sections below illustrate how we designed each of these components of the walker system.
Walking mechanism
The walker has two toeholds (overhangs) on either end. These represent the “legs” of the walker. The walker with bind to corresponding toeholds on the first two hairpins (aA, bB) of the track. This is the starting point of the system. When the walker binds to the track, the hairpins will open exposing a domain that was inaccessible. The fuel F1 binds to this toehold and displaces the lagging leg. This leg will then bind to the next hairpin in line, cA, as it did with the first, now becoming the leading leg by hopping over the other. This is the first step.
The duplex formed by aA and F1 expose a binding domain specific to the second hairpin F2.F2 then displaces F1 from aA, forming a duplex F1:F2. This duplex is free-floating in solution. Because of this it can bind to bB and displace the now lagging leg, allowing it to bind to dB. Now the walker has taken a full two steps.
Finally a nicking enzyme (name it) specific to a domain formed only when F1:F2 is bound to a hairpin with a B loop will cut the formation. This cut causes the total number of bonds in the duplex to decrease to the point in which they can freely dissociate from the track [cite], becoming waste. This will allow the hairpin bB to return to its initial state, thus regenerating the track.
This process can continue as the track is lengthened and as long as there is fuel available for consumption. However our design incorporates elements that limit the length of the track. Almost all things observed in nature tend to follow exponential curves. This is no different in the reaction times for DNA strand displacement. We know from observation that not all walkers will complete their first steps at the same time, and some may not even complete one. This is due to the subtle differences in free energies of the displacements that our design employs. Because the drive is not very strong, we expect the walkers to decrease in number as they continue moving forward due to energy dips and mismatched base pairs.
Fueling the walker
Directional walking cannot be achieved without a source of energy, such as a fuel molecule [cite Kelly 2005]. The fuel molecules we designed transmit energy to our system via the CHA (Catalytic Hairpin Assembly) reaction [cite].
BB: Andy, please copy the contents of CHA-kinetics.docx here.
We built a bipedal walker by hybridizing two strands of DNA that have overlapping domains on both sides.
The walker utilizes a track that is composed of eight strands of DNA. Two make up the base of the track while the other six are hairpin structures (DNA that partially binds to itself) that are hybridized to the two base strands forming a long duplex with alternating hairpins along its length. These hairpins will serve as the footholds for the walker to attach itself to. The walker is driven to walk by repeated instances of the CHA reaction and a series of toehold-mediated stand displacement reactions. After the walker has taken two “steps” the second hairpin on the track will have a long duplex that a nicking enzyme will cut specifically. This will cause both pieces to dissociate from the track due to a lack in strength of binding. Because these fuels are added once to the system at the same time, and are removed from the track by a nicking enzyme, the walker is both autonomous and non-destructive to the track.
Why is the above text here???
Acknowledgements
We are sponsored by Integrated DNA Technologies (IDT, Coralville, IA, USA). We received travel support from the College of Natural Sciences, the University of Texas at Austin.
Background photo taken by Linhao Zhang in Fort Davis, Texas. Used with permission.
