spms01 developing rna binding peptide nucleic … oral...1 solid phase peptide synthesis using...
TRANSCRIPT
SPMS01Developing RNA Binding
Peptide Nucleic Acids
Contents1. Introduction of the background 2. Hypothesis 3. Methodology4. Results and Discussion 5. Conclusion 6. Future work and Applications
Introduction
➔ A chronic neurodegenerative disease
affecting approximately 29.8 million
people worldwide.
➔ This disease presents a characteristic
early symptom of short term memory
loss.
Alzheimer’s
A quick summary!
Extrapolate to greater potency and less side effects for both Alzheimer’s and other neurodegenerative disease applications.
Develop a promising way to tackle Alzheimer’s through the synthesis of PNAs complementary to the tau pre-mRNA.
A novel binding mechanism that targets both duplex and single regions in the pre-mRNA
Enables reduction of the isoform ratio by exploring the therapeutic applications of the PNA
Value-adds to the field of research
Tau Hypothesis
➔ Hyperphosphorylated tau proteins pair
up and form neurofibrillary tangles
inside nerve cell bodies.
➔ The cell microtubules disintegrate,
destroying the structure of the cell's
cytoskeleton which collapses the
neuron's transport system.
Hypothesis
Binding affinity of the PNA strands with the RNA would be higher when manually synthesized
monomers such as L, Q and E are used and when they are both single and double stranded resulting
in the formation of both duplex and triplex structures.
Tau Hypothesis disintegration of microtubules which disrupt neuron cell transport system
Methodology
11 Solid Phase Peptide
Synthesis
2Purification of PNAs
33 Polyacrylamide Gel
Electrophoresis (PAGE)
Methodology
➔ Synthesis of two PNA strands, with sequences NH2-Lys-TAAAAALTLT-CONH2 and NH2-Lys-GGACLELELT-CONH2
➔ PNA monomers are supported by solid methyl benzhydrylamine hydrochloride polystyrene (mBHA) based resin with Boc Protecting group
➔ Wash resin with dichloromethane (DCM) and dimethylformamide (DMF) repeatedly with exposure to nitrogen gas
➔ Add coupling solution ➔ Perform Kaiser's test➔ Neutralisation➔ Repeat procedure manually for synthesis of the entire strand
1 Solid Phase Peptide Synthesis
➔ Using RP.HPLC➔ PNA oligomer was first cleaved from the resin ➔ PNA oligomer that precipitated out was then dissolved in water
and purified using double RP.HPLC so that the purest samples are used in PAGE
2 Purification of PNAs
3 Polyacrylamide Gel Electrophoresis (PAGE)
➔ Gel was placed at 4 degree celsius for 5 hours overnight➔ The gels were then stained in ethidium bromide for 30 mins➔ Visualisation using Typhoon Trio Variable Mode Imager. ➔ Testing of the binding affinity of the PNA strands with their
complementary double stranded RNA molecule
Results and discussion
Results and Discussion
Lower lane
Upper lane Second RNA First RNA
A decrease in the 4-repeat/ 3-repeat (4R/3R) isoforms
2
Exon 10 inclusion during splicing
1
The formation of neurofibrillary tangles are significantly inhibited.
3
The highly
successfuland
remarkablebinding shown on the gel indicates a similar binding mechanism for
the tau pre mRNAs and the
PNAs.
Prevent gain of function mutation: Hairpin loop is destabilised to form a single strand with greater accessibility to splicing elements
such as the U1snRNP.
Future work and applications
➔ Pave way for DNA silencing using
duplex PNA.
◆ Since the binding affinity is
an important factor affecting
the targeting and inhibition
of the RNA.
➔ There is a great potential with
regards to the production of such
drugs due to the higher possibility
of mass production with less
contamination as compared to
antibodies
ApplicationsFuture Work
➔ The actual effect and impact of the
PNA strands in the cellular setting
has to be tested.
◆ Thus, cell culture studies are a
must to test the actual effect
of the synthesized PNA on the
cells.
➔ The exact ratio between the 4R
and 3R isoforms can then be
identified to assess the significance
of our solution.
Conclusion
On the whole, for this project, we
have focused on the increasing of
the binding affinity of the PNA with
the target through the
development of a new duplex and
triplex binding system and have
proven the increase in the binding
as well.
● Alzheimer's disease. (2018, December 08). Retrieved from https://www.mayoclinic.org/diseases-conditions/alzheimers-disease/symptoms-causes/syc-20350447 ● Toh, D. K., Patil, K. M., & Chen, G. (2017). Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified
Peptide Nucleic Acids. Journal of Visualized Experiments, (127). doi:10.3791/56221 Retrieved from: https://www.ncbi.nlm.nih.gov/pubmed/28994801● (Gupta & Tomar R., 2012): Antisense Technology In Kumar, Gupta & Mohan (Ed.) Biotechnology in Medicine and Agriculture Principles and Practices ( pp 297-312 )
Thank you
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