lecture 3: RNA inhibition

Why do RNA inhibition

: RNA inhibition is a way scientists control how genes work by blocking specific RNA molecules. This helps in studying genes and can be used to treat diseases

Three strategies to RNA inhibition and blocking protein synthesis

o DNA oligonucleotides (ODN)

o Antisense RNA (ASO)

Triple helix forming oligonucleotides (TFOs

What are DNA oligonucleotides?

short sequences of DNA or RNA. They can be designed to bind to specific genetic sequences, allowing control of how genes function.

Using One Short DNA Oligonucleotide

A single short DNA strand can be used to sequence DNA

Using two short DNA oligonucleotides

· Two short DNA sequences can be used in Polymerase Chain Reaction (PCR) to amplify (copy) DNA, which helps in genetic testing, disease detection, and forensic science.

Antisense oligonucleotides (ASO)

single strands of DNA or RNA that are complementary to a chosen sequence

Once ASO binds to mRNA it can cause

inhibition of 5'- cap formation and 3' poly-a-tail

inhibition of RNA splicing

activates RNase H to degrade mRNA in the nuclei and cytoplasm, hinderance of ribosomal subunit binding

modification of 1st gen: O to S in phosphodiester bond renders it

· more resistant to RNase H, inc. bioavailability and inc. solubility

modification of 2nd gen: + -CH3 or -O-CH3 @ Oxygen 2' position renders it ·

· less toxic and enhances affinity for RNAs)

modification of 3rd gen: adding furanose to the base (a LNA or PMO) to make it

· nuclease resistant, inc binding affinity and inc biostability

The template/noncoding strand

antisense DNA strand used as a template for RNA synthesis

coding strand

the strand of DNA that is not used for transcription and is identical in sequence to mRNA, except it contains uracil instead of thymine

what are Triple helix-forming oligonucleotides

Normally, DNA has two strands (double helix), but a third strand can bind to form a triple helix. (Hoogsteen base pairs)

function of TFOs

o TFOs block genes from being turned on, preventing cells from making specific proteins.(blocks the binding of TFs to a specific DNA sequence)

Hoogesteen base pairing

an alternative form of base pairing in DNA or RNA where the hydrogen bonds between the nitrogenous bases deviate from the conventional Watson-Crick base pairing.

Conventional Watson-Crick Pairing

Normally, adenine (A) pairs with thymine (T) in DNA (or uracil in RNA), and guanine (G) pairs with cytosine (C), following the classic structure.

Hoogesteen base pairing examples

Adenine (A) can form a hydrogen bond with thymine (T) in a different orientation compared to the Watson-Crick pairing.

Guanine (G) can form a hydrogen bond with cytosine (C) or thymine (T) in an alternative configuration.

two types of TFOs

Intermolecular

intramolecular

Intermolecular TFOs

the triplex structure forms between a single TFO and a separate, double-stranded DNA molecule.

Intramolecular TFOs

the triplex structure forms within a single DNA or RNA molecule, where a TFO sequence interacts with another region (homopurine and homopyrimidine) of the same molecule.

transcription factors

Collection of proteins that mediate the binding of RNA polymerase and the initiation of transcription.

Transcription factor decoys

create decoys (fake binding sites) to trap these transcription factors and stop them from turning genes on

drawback of TF decoys

o However still some issues with purity, stability and transfection issues with decoys

o Comes with limitations

RNA Inhibition Can Happen in Two Ways

§ Skipping exons during transcription

§ Blocking mRNA from Making Proteins

siRNA is made in

Made in a lab and inserted into cells.

miRNA is made in

Naturally made by cells and can control multiple genes.

siRNA process

Dicer cleaves/ unwinds double stranded mRNA into siRNA.

there is two strands the guide and passenger strand (Passenger strand is degraded)

Guide siRNA direct Ago in RISC and binds to mRNA perfectly

the RISC bind the mRNA in two ways

fully complementary and partially complementary

fully complementary binding

leads to target cleavage and mRNA degradation(siRNA)

partially complementary binding of RISC

recruits cofactors (miRNA)