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

All images self taken unless otherwise stated