DNA Synthesis in Space

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SSI Biology leaders hosting a workshop, taken by Lillian Zhu

Space is modern science’s largest laboratory, and the record of innovations and breakthroughs that have been made in space or through programs focused on space is incredible.  Technological development and scientific research have benefited immensely from out-of-this-world discoveries, but biology has a significant drawback compared to most disciplines. Synthesizing DNA, a molecule crucial to the existence of all organisms, is currently impossible in space. Any experiment requiring DNA synthesis is locked out of the largest laboratory in existence. However, Stanford’s Student Space Initiative has made breakthroughs on promising solutions that might provide the key to making DNA synthesis more accessible.

Synthesizing DNA is a crucial first step in a technique called PCR, or polymerase chain reaction. This process takes a small segment of sample DNA, which is virtually undetectable, and replicates it to create millions, billions, or even more strands. DNA can be amplified through PCR because of its unique molecular structure. Subunits of DNA called nucleotides can have any of four nitrogenous bases attached to them: adenine (A), cytosine (C), guanine (G), or thymine (T). Since DNA is double-stranded, each nucleotide is paired with another on the opposite strand. Crucially, these pairs are not random. Adenine and thymine always pair together, and cytosine and guanine must always match up. Each section of DNA on one strand can only be matched with one specific sequence of bases.

In order to identify the exact sequence of DNA to be replicated through PCR, a short segment of DNA called a primer must be synthesized. If the sample has a matching sequence to pair with the primer, the primer can attach to it. The primer is able to serve as a basis for the replication by starting the second strand of the DNA. In about an hour, a very small amount of sample DNA can be exponentially copied to allow for detection and examination.

The ability to identify DNA from trace samples is useful in a variety of disciplines. For example, PCR could be used to discover the guilty party among several suspects, making it a useful tool in forensics. Ecologists can use it to detect the presence of a pathogen in water. In medicine, PCR can be used to determine the presence and identity of microbial species or disease-causing changes in DNA. But before PCR can be carried out, the correct primers must first be created through DNA synthesis.

While DNA synthesis is a process that occurs naturally in the body using catalysts called enzymes, the DNA synthesis that is conducted in a laboratory often uses a series of chemical reactions called the phosphoramidite pathway. This method is the most common process used in industrial settings because it is efficient and relatively cheap. The industries that use DNA synthesis have also had plenty of time to optimize the process, which was developed in the 1980s. In many settings, such as hospitals in developed countries, the pathway can be implemented easily.

Developing countries and hard-to-reach areas usually lack the required infrastructure for this process, which involves toxic chemicals both as starting points and byproducts of the reaction. In developed countries, hospitals already have the facilities needed to address the toxic waste. However, in other areas, shipping and disposing of the needed materials is impossible.  Consequently, researchers and citizens in developing countries do not have access to certain methods of research or medical treatments. While the number of researchers who would require a different method of DNA synthesis is not large compared to those using traditional methods, the work they do could still have wide-ranging impacts. But since the number of affected researchers is small, companies have little incentive to address these problems and the available funding for these areas is limited.

To address these problems, Stanford’s Student Space Initiative, or SSI, has been working for two years on creating an efficient and portable alternative to the phosphoramidite pathway.  Ultimately, the organization hopes that this technology will be available in space.  The goal is to produce a transportable, printer-sized device that can synthesize DNA efficiently, using a different set of enzymatic reactions.  All involved chemicals are water-soluble, which eliminates the problem of toxic waste and makes research possible in developing countries or remote locations.

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SSI Biology Team running experiments, taken by Alan Tomusiak

The current method that the group is using was originally conceived in the summer of 2017, about a year after the project launched.  It uses the TdT enzyme, already found in the human body.  TdT’s natural function is to randomly add nucleotides to DNA, which serves the purpose of generating pathogenic DNA to train the body’s immune system to respond. When it comes to synthetic DNA, the TdT enzyme is a promising, non-toxic way to extend a DNA strand from a short primer sequence.

Unfortunately, the TdT does not stop after a single addition of the nucleotide.  For example, if the primer was GCTTTACTG, and next nucleotide to be added is adenine, the result would not be GCTTTACTGA.  It would be GCTTTACTGAAAAAAAAAAAAAA…, which is not the desired sequence. SSI is investigating two different solutions to address this problem, one preemptive approach and one that backtracks to delete the undesirable repeats.

The preemptive approach adds a special type of modified nucleotides, changed in a way such that the chain stops after adding just one copy of the desired nucleotide.  In this case, using the previous example, and an asterisk to denote modification, the primer GCTTTACTG would be added to a mixture containing the TdT enzyme and adenine*.  The result would be GCTTTACTGA*, and the modification of the nucleotide can then be reversed to produce GCTTTACTGA.  SSI has successfully used this method to add a nucleotide to a strand of DNA and block further additions.

There’s also a retroactive approach that SSI calls the “backspace method.”  It uses another enzyme to cut off the extra nucleotides that TdT added beyond the single addition.  This method is not perfect yet, however. Ironically, like TdT, this enzyme also doesn’t know where to stop. Rather than just removing the extra nucleotides as intended, it could cut the whole strand apart. SSI has been testing a solution to this problem for the past two months. Three weeks ago, the members of the group ran a test to prove that this concept is viable. They extended a segment of DNA that was 20 nucleotides long to one that was only 21 nucleotides long.

Once SSI works out the details on what synthetic mechanism might provide a viable alternative to the phosphoramidite method, the next challenge is to design a device that can carry out this mechanism. One option involves a significant amount of tubes to introduce chemicals and remove waste. The tubes would operate similarly to heart valves and direct all chemicals to the appropriate locations. However, the requirement for such a large amount of tubing has led SSI to explore alternative options.

The current design for a device to carry out PCR involves a process called electrowetting.  A printed circuit board is covered by several pads, which are covered by a hydrophobic, or water-repelling, surface. A water drop is placed between the hydrophobic surface and the pad, and the water is drawn into the pad due to the surface’s repulsion of the water. A high voltage of electricity, approximately 300 volts, can be transmitted through the circuit board to move the water to different pads throughout the system. Chemicals can be manipulated across pads using electricity, so it is possible to control the location of each chemical and to mix them appropriately without all of the tubing required in the other design. A magnet placed on the bottom of the printed circuit board would hold DNA in place.

Using electrowetting and the backspace method for DNA synthesis, SSI has developed promising alternatives to the traditional phosphoramidite method. Currently, both techniques are undergoing development and testing and have shown success already. The next steps for the group include further testing to ensure that all procedures function well and improving efficiency. Once individual components are tested, transferring the DNA synthesis to the device will be the next development. In the end, the team hopes this device could be used for remote research, hospitals in developing countries, and even research in space.

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SSI Biology team during a research paper reading session, taken by Alan Tomusiak

References

  1. Tomusiak, Alan. (26 February 2018). [Personal Interview]
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