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The last time I spoke to you, I introduced you to a rather promising method of DNA origami (3 dimensional DNA shapes) which already has a range of applications. In my previous article, I outlined the use of DNA “boxes” in a case study where DNA Boxes appeared to successfully deliver chemotherapy drugs to tumour cells, resulting in tumour shrinkage without the unpleasant side effects commonly associated with chemotherapy [1]. Now a recent paper published in Nature by Benson et al [2] described an even easier method of assembling DNA Nanostructures shapes, perhaps ushering in another wave of increasingly sophisticated computational and medicinal techniques at minimal cost.
It seemed like a good time, therefore, to summarise recent advances in DNA origami since I last visited this topic. Choosing which studies to cover came with its own challenges since I wish to tell you about them all, but for the sake of sanity I have chosen two which I have found particularly fascinating.

 

Experiment 1) DNA Origami in cancer detection

I have previously discussed the potential for DNA origami boxes to deliver therapeutic cargos to target areas of the body where they are most needed. However, great potential seems apparent not only in the treatment of, but also in the early diagnosis of cancer. This is a good time to introduce the Liu et al [3] and their work on MicroRNAs: small pieces of genetic material which have no coding function but act as regulator tools within the body. Some, like hsa-miR-21, have been linked with the onset of lung cancer. However, detecting hsa-miR-21 presents a challenge in that it is a) very, very small and b) exists in small amounts. Thus the ideal solution would be to construct a machine that is highly sensitive to these specific MicroRNAs and it seems that DNA origami provides that very solution.

The Machine:
The answer to the challenges above came in the form of what looks, for all intents and purposes, like a birthday cake. But in fact, this electrochemical sensor, the Genosensor, was designed with a unique DNA origami “Loop” component which is specific to hsa-miR-21 while a ferrocene tag provided signal reading during the experiment (Fig.1).

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What was found?

1) The genosensor was successfully able to detect trace amounts of hsa-miR-21. This was made evident by the results indicated in Fig.2, where a clear linear relationship can be observed. The line shows increasing signal output correlating
cells of the cremaster muscle at three time intervals. Image from [4].Figure 1) Assembly of the genosensor probe, indicating “bending mechanism” caused by hs-miR-21 binding (E). Image modified from [3].
with an increase in MicroRNA concentration. This suggests that the genosensor is responding appropriately to the MicroRNA target.

2) The tetrahedral shape of the genosensor proved the ideal shape for MicroRNA binding and detection. The authors noted that tall, open conformation of the probe allowed for the targeted MicroRNA to bind easily. Once bound, the Stem-loop DNA at the top of the probe would contact the base of the probe, activating a signal which can then be detected. This is promising as it demonstrates how a clever design can aid in sensitive detection of MicroRNA.

3) The genosensor was able to function in a cellular environment. This was made apparent by the similarity of the two curves shown in Fig.3- which shows activity in a cell and non-cell environment. However, it is important to note that cell lysate (a gooey substance made of scrambled cells) may not be representative of how cells may behave when alive with fully working compartments. Furthermore, there is no information concerning repeats of this aspect of the experiment, therefore caution is advised when assessing the validity of this claim.

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In summary…

The study outlined above describes hopefully the first of many such sensors to come as the applications of DNA Nanostructures becomes increasingly easier and cheaper over the next few years. The genosensor probe in this instance shows promise as a diagnostic tool; however I would like to see future research on its ability to operate in vivo as this will provide information on the probe’s ability to withstand some of the many surprises that the human body no doubt has in store.
Now before I go on….
It would seem at this point, the Jury is still out on the ability for DNA Origami to successfully operate in vivo considering, at least in my opinion, the lack of statistically significant evidence. However there may still be hope as I managed to stumble across this intriguing article. It took me a few attempts and copious mugs of tea before I got the gist of it, but in the end it looks like an idea I could get behind.

 

Experiment 2) DNA Origami-the Noble Steed.

In light of the myriad problems involved with introducing foreign objects into the body, a study investigated the use of DNA origami (in the form of nanotubes) in the delivery of special sequences termed “CpG sequences” which can aid in the immune response [4]. An example of this aid could be when negating the immunosuppressive activity of AIDS or certain cancers.
CpG sequences exist naturally within two states: methylated (a specific chemical change) and unmethylated (no chemical change). Unmethylated CpG exist within bacterial DNA (such as might be expected during an infection) and stimulate proteins such as Toll-Like Receptor 9 (TLR9) which triggers inflammation (something that we all know looks ugly and painful but it is in fact an ingenious form of biological defence).
So why DNA origami? Well, since DNA is inert within the body, it reduces the risk of nasty side effects in a patient. CpG sequences and DNA origami therefore make a potentially ideal Knight-and-Steed in which to direct the immune system’s machinery where it is required (Fig.4).

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What was found?
1) Did CpG carrying DNA nanotubes localised into surrounding tissue of the injection site? Yes. This is important as it addressed question as to whether the DNA nanotubes would simply disperse into the body, potentially causing a range of side effects. However, Fig.5 shows internalisation into nearby perivascular tissue resident cells.

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2) Did the CpG-carrying DNA nanotubes increase leukocyte recruitment? Yes. This is apparent from Figure 6. This suggests that the effective delivery of CpG tails made possible by DNA origami can initiate a meaningful immune response.

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3) Nf-kB pathway was activated in surrounding cells. Nf-kB is a protein which mediates immune response and part of its structure includes Phospho-p65. The activity of this pathway was indicated by that fact that all the cells surrounding the injection site became positive for Phospho-p65 (Fig.6). Like result No.2, this testifies to the efficacy of DNA origami in this study.

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In summary….
This particular study may not have DNA origami as the star of the show; however it demonstrates that DNA nanostructures can play a pivotal role in the treatment of diseases which, by nature, aim to suppress the immune system of a patient.

 

Concluding thoughts:

So there we have it: the two studies discussed here suggest a promising and inventive future for DNA origami in the realms of medicine. Furthermore, such a future of new and powerful treatments is only made even easier to accomplish with the advent of easier construction methods such as that outlined by Benson et al [2]. The promises of DNA origami is not only the preserve of medicine however, as even a perfunctory search on the topic will provide a mountain of advances in computing and engineering too. It is therefore likely that we soon be witness the golden age of DNA nanotechnology and I invite you in joining me as I keep an eye open for more advances.

 

References:

[1] Zhang, Q., Jiang, Q., Li, N., Dai, L., Liu, Q., Song, L., Wang, J., Li, Y., Tian, J., Ding, B. & Du, Y. (2014). DNA Origami as an In Vivo Drug Delivery Vehicle for Cancer Therapy. Acs Nano 8, 6633-6643
[2] Benson et al, (2015), DNA rendering of polyherdral meshes at the nanoscale, Nature, 523, pp441-444.
[3] Liu, S., Su, W., Li, Z., Ding, X., (2015), Electrochemical detection of lung cancer specific microRNAs using 3D DNA origami nanostructures, Biosensors and Bioelectronics¸7, pp57-61.
[4] Sellner, S., Kocabey, S., Nekolla, K., Krombach, F., Liedl, T., Rehberg, M., (2015), DNA nanotubes as intracellular delivery vehicles in vivo, Biomaterials, 53, pp453-463.