Biomod/2013/IIT-Madras/Project

Background

DNA walkers are nanomachines which exhibit linear motion along a DNA strand. They ware initially constructed with the aim of mimicking the intra-cellular motion of molecular motors such as kinesin so that they could in turn serve as mechanical parts for future Nano-bots. Their structure mainly consists of two strands - a moving strand and a stationary strand along which the moving strand "walks". The major components of the DNA walker system are[1][2]:

1. Walker : The moving strand
2. Track : The stationary strand
3. Attachment strands/Set strands : These hybridize to single stranded segments on the walker and track strands thereby linking them.
4. Detachment strands/Unset strands : They have high affinity for the set strands and displace them via toehold mediated strand displacement.

There have been several modifications to this construct depending on the strategy of actuation used[1][2][3][4][5][6][7].

In this project, we demonstrate the displacement of DNA in response to stimuli such as pH change.

History of DNA Walkers

DNA Walkers in the past have primarily used other synthetic DNA oligonucleotides[1][2] in order to exhibit motion over a track. Other actuation strategies include the use of light[6][7] and restriction enzymes[4][3].

These systems have been extremely effective in controlling motion along a track. However, they may not be able to function in the endogenous cell environment ie. without the addition of "Extra" synthetic biomolecules.

Idea!

In an attempt to overcome this, we embarked upon designing a DNA machine that responds to changes in cell environment by "hopping" along the track.

We intend to control the DNA Walker using

1. pH
2. Light - We have used a short DNA Strand as a proxy for caging the DNA with photo-activatable groups[8]

Resting State of DNA "Hopper"

Module I: pH Switch

This Switch exploits the formation of intra-molecular i-motif at lower pH[9]

The mismatches required in the input strand (I1) is calculated based on the Gibbs free energy of i-motif formation and input (I1) -stator (L1) hybridization energy.

1. Module II: short complementary DNA (Proxy for caging DNA with photo-activatable compounds)

This ssDNA Strand can be caged at dA and dC residues by NDBF (photo-activatable groups). Due to the un-availability of photo-activatable caged DNA, we have caged DNA strands using a short stretch of complementary DNA.

Final Structure of DNA Walking Platform

These modules are linked to form the DNA Walking Platform.

DNA "Hopper" in motion

At Low pH (pH<5.5), L1 folds into an intra-molecular i-motif due to the formation of CHC+ (hemi-protonated Cytosine base pairs). This folding doesn't release the Input (I1) into the solution, but assists toehold mediated strand-displacement to module 2.

Module 1

Module 2

Complete Picture :: DNA "Hopping"

Translation of Input (I1) from Module 1 to Module 2 triggered by pH (change in cellular environment)

Refernces

1. A Precisely Controlled DNA Biped Walking Device William B. Sherman and and Nadrian C. Seeman Nano Letters 2004 4 (7), 1203-1207 [1]

2. A Synthetic DNA Walker for Molecular Transport Jong-Shik Shin and and Niles A. Pierce, Journal of the American Chemical Society 2004 126 (35), 10834-10835 [1]

3. Yin, P., Yan, H., Daniell, X. G., Turberfield, A. J. and Reif, J. H. (2004), A Unidirectional DNA Walker That Moves Autonomously along a Track. Angew. Chem., 116: 5014–5019. doi: 10.1002/ange.200460522

4. Bath, J., Green, S. J. and Turberfield, A. J. (2005), A Free-Running DNA Motor Powered by a Nicking Enzyme. Angew. Chem. Int. Ed., 44: 4358–4361. doi: 10.1002/anie.200501262

5. Molecular Gears:  A Pair of DNA Circles Continuously Rolls against Each Other Ye Tian and and Chengde Mao Journal of the American Chemical Society 2004 126 (37), 11410-11411[1]

6. Building a Nanostructure with Reversible Motions Using Photonic Energy Mingxu You, Fujian Huang, Zhuo Chen, Ruo-Wen Wang, and Weihong Tan ACS Nano 2012 6 (9), 7935-7941 [1]

7. You, M., Chen, Y., Zhang, X., Liu, H., Wang, R., Wang, K., Williams, K. R. and Tan, W. (2012), An Autonomous and Controllable Light-Driven DNA Walking Device. Angew. Chem. Int. Ed., 51: 2457–2460. doi: 10.1002/anie.201107733

8. Wavelength-Selective Uncaging of dA and dC Residues Florian Schäfer, Khashti Ballabh Joshi, Manuela A. H. Fichte, Timo Mack, Josef Wachtveitl, and Alexander Heckel [1]

   Organic Letters 2011 13 (6), 1450-1453