Conclusion & Discussion

Conclusion

As we anticipate, the structures and functions of NanoNeedle should have the following characteristics

A. Structures that we design:

• 1. NanoNeedle folds into hexagonal prism and forms the enclosed top with mesh structure.
• 2. The weak structure is able to bend when attaching to the cell wall of bacteria; the strong structure is tough and long enough to penetrate through bacteria.
• 3. The plasmid should be closely bound inside and stably exist before NanoNeedle contacts with the target bacteria.

B. Three functions that we expect:

• 1. Recognize target bacteria specifically without acting on human cells.
• 2. Penetrate through the cell wall and membrane and form a pore on cell membrane for the plasmid delivery.
• 3. Deliver the plasmid into the cytoplasm of target bacteria.

Discussion

A. Our experiment design and results

• 1. The reason why we chose the GFP gene instead of suicide genes as our target gene.

  We use the GFP gene to confirm whether NanoNeedle can work and actually deliver plasmid into the bacterial for expression or not. If we place suicide genes into NanoNeedle and bacteria die after incubation, we cannot confirm that bacteria accept the plasmid from NanoNeedle and are killed by expression of suicide genes. Thus, it is difficult for us to check if bacteria destroy their own physiological functions by encoding proteins. Another possible reason might be too many pores formed by NanoNeedle causing cell lysis.As a result, we chose the GFP gene as our tool for confirmation.

• 2. Result of TEM imaging

  The stained rectangular prism, our NanoNeedle, is about 123nm in length and 25nm in width under TEM. Nevertheless, the estimated scale of NanoNeedle we predict is 100 x 20nm. It shows that the structure is a little larger than we expected. We predicted the possible reason is that the base-pairings between staples and scaffolds are not perfectly formed as double-helix structure with radius of 1.1 nm. After the plasmid is bound to aptamers inside NanoNeedle, the plasmid may expand the radius of the whole structure.

  Also we unexpectedly found that there are patterns of impurities in the view of TEM. These impurities might be precipitation caused by the acid-base neutralization between the acidic stain we used and the slightly basic buffer for NanoNeedle.

• 3. the reason why the result of cell culture cytotoxicity showed the sign of proliferation of HT29.

  The possible reason could be the fact that the structure of NanoNeedle is made of DNA. Through endocytosis it can be transferred into the cell and broke down into nucleotides as nutrients.

B. Future work

NanoNeedle is a versatile tool to fight against various antibiotic-resistant bacteria by changing aptamers and plasmids with different genes. Therefore we plan following experiments that we have not yet performed due to the time limitation.

• 
  1. The process of NanoNeedle acting on bacteria under TEM imaging.

  In the functional test, we proved that NanoNeedle perfectly performs its three functions on bacteria by detecting GFP signal. However, seeing is believing, we would like to find other convincing evidences by using TEM to capture the moment that NanoNeedle binds to the cell wall of target bacteria.

• 
  2. Manipulate aptamers to recognize different bacteria

  We will continue to find other DNA aptamers showing high affinity and selectivity to anti-resistant bacteria. For example, we use aptamers targeting “Pseudomonas aeruginosa, a multiple antibiotic-resistant bacteria causing cross-infections in hospitals. Also we would like to target methicillin-resistant Staphylococcus aureus (MRSA), a bacterium that is resistant to many antibiotics and responsible for several difficult-to-treat infections in humans.

• 
  3. Change target genes

  After we prove that NanoNeedle can transfer plasmid into bacteria, we would like to send suicide genes into target bacteria to check if they can kill bacteria as we expected. This experiment will verify whether NanoNeedle can treat pathogens in the human body for clinic applications.

  In the near future, we believe that NanoNeedle will become an alternative therapy for antibiotics, achieving our final goal, effectively fight against many pathogenic antibiotic-resistant bacteria in human body.