What is antisense?
Antisense are generally shorter fragments of nucleic acid that are complimentary to the sense strand of the mRNA
Sense mRNA codes for protein translation.
Therefore, antisense strand = ______ strand
We can use antisense mRNA to bind to sense mRNA to regulate its ____1____ and ____2____. Turn on and off.
Regulation of eukaryotic gene expression
1, 2, 3 & 4 = points of ______
What does capping do?
Capping = adding a 5’ methyl-guanosine to the mRNA
- Helps to stop mRNA from being degraded by ribonucleases
- Acts as a site for initiation of translation
What is splicing?
The removal of introns to produce the mature RNA to be translated.
What does polyadenylation do?
Polyadenylation = leads to cleaving of the protein at the 3’ site and adding of a polyadenosine tail.
It stabilises the mRNA.
What is gene editing?
Gene editing = single amino acid insertions, deletions, mutations etc.
Describe nuclear export.
Nuclear export = The transport of the mature mRNA from the nucleus to ribosomes.
Introns and exons can be any length, therefore we need a mechanism to enable our splicing machinery to know where to cut.
This mechanism are the 5’ and 3’ ______ ______ ______.
Conserved splice sequences
Describe the two-step splicing of introns.
- Cut at 5’ splice site
- Creation of bond between 5’ end of intron and branch site
- Cut at 3’ splice site to release intron lariat
- Ligation of two exons
Naturally, antisense prevents the ____1____ machinery from binding to the mRNA thus blocking (turning off) the ____2____.
What causes it?
Called Mediterranean anaemia. Essentially a blood disorder where the haemoglobin is not processed properly. Leads to all sorts of anaemia like symptoms.
Caused by cryptic splicing.
What is a cryptic splice site?
How can we treat this?
Usually unused splice sites but if we get mutations in our gene, these cryptic splice sites can be activated. These leads to the translated protein being prematurely spliced, leading to a truncated protein being produced.
To prevent this we can use antisense RNA that will bind to the cryptic splice site mRNA thereby blocking it and prevent its translation.
Duchenne Muscular Dystrophy
A C→T mutation causes a stop codon to be present in the middle of the gene. This leads to a ____1____ protein (dystrophin) being produced in sufferers. To eliminate the symptoms of Duchenne Muscular Dystrophy we can use ____2____ RNA to block this UGA (stop) codon and the exon it resides in thereby allowing a shortened dystrophin to be produced. It may be shortened but it still has greater functionality that the ____1____ form as exon 23 was found to not be vital for the proteins activity.
Spinal Muscular Atrophy is a wasting disease where the neurons don’t form properly. Often fatal to infants.
Sometimes for splicing we need enhancers.
In exon 7 of the SMN protein there is an exonic enhancer of splicing.
SF2/ASF = Splicing Factor 2 / Alternative Splicing Factor
In normal SMN, SF2 binds to the splice site and initiates translation.
In the disease, there is a mutation in the enhancer leading to the splice site being skipped. This leads to exon 7 not being translated. A truncated -6,8- protein is produced = unstable SMN = disease phenotype.
So how can we treat this?
With antisense, we can remove the splice site protein and attach it to a piece of antisense RNA which exactly matches the mutated enhancer site. The protein is then put back into the system where it integrates back to where it originally was.
Splicing can then occur as normal and the normal SMN protein is produced = no disease phenotype.
List the problems with antisense. (3)
- RNAs are relatively unstable (attacked by ribonucleases etc.)
- Difficult to transfer into cell
- Natural mechanisms in the cell block antisense function or degrade
Normal DNA = Base (B), Deoxyribose sugar and phosphate backbone. Can be degraded – is usually degraded at the phosphate-sugar bond.
But we can fool the cell machinery into not degrading it by producing synthetic oligonucleotides by replacing a part of the ____1____ or ____2____ groups. This stops the cell from recognising it as DNA, thereby preventing its degradation.
RNA, that is more unstable due to having a 2’ OH group instead of a 2’ hydrogen on the sugar group. This enables self-cleavage and therefore degradation. So what we can do with synthetic RNAs is to change the 2’ OH group, for e.g. normally to a ____3____ or ____4____ group.
What are the benefits of modified nucleic acids? (3)
- Modifying the sugar phosphate backbone reduces the rate of turnover
- Modification results in molecules that are easier for cellular uptake
Not recognised as nucleic acids
- Important as the cellular machinery doesn’t sequester the antisense
What is a ribozyme?
The hammerhead ribozyme
- Naturally occurring ribozyme that is present in several ____1____ RNA viruses
- Used to cleave individual genomic RNAs during rolling circle ____2____
- Naturally a cis-cleavage but can cleave in trans
We can make ribozymes that don’t have to self-cleave. We can produce ____1____-ribozymes that can cleave things other than themselves. We can do this by designing the RNA ribozyme to bind to the specific target site that we wish to cleave (e.g. pathogenic RNA virus). The ribozyme binds through the ____2____ of antisense. When bound, the ribozyme forms its shape, then the active site is able to interact and cleave the target sequence.
An aptamer is effectively an artificial ______.
When using SELEX to synthesize DNA aptamers, how can we separate the dsDNA to ssDNA?
Why does it need to be separated?
To ensure this, one of the primers contains a biotin molecule at its extremity. We can then pull the biotin down on streptavidin beads to separate out the ssDNA with the biotin on by magnetically separating it from the rest of the DNA that hasn’t got the biotin on. This leaves us with ssDNA.
dsDNA cannot form the secondary structures of interest.
Define RNA interference (RNAi).
A biological process in which RNA molecules inhibit gene expression.
Define small/short interfering/inhibitory RNA (siRNA).
A class of double-stranded RNAmolecules, 20-25 base pairs in length. siRNA is essentially a synthetic, man-made, miRNA molecule, and operates within the RNA interference (RNAi) pathway, where it interferes with the expression of specific genes with complementary nucleotide sequences by degrading mRNA after transcription, resulting in no translation.
Define microRNA (miRNA).
A small non-coding RNA molecule (containing about 22 nucleotides) found in plants, animals and some viruses, that functions in RNA silencing and post-transcriptional regulation of gene expression.
The RNA-induced silencing complex, or RISC, is a multiprotein complex, specifically a ribonucleoprotein, which incorporates one strand of a double-stranded RNA (dsRNA) fragment, such as small interfering RNA (siRNA) or microRNA (miRNA). The single strand acts as a ____1____ for RISC to recognize complementary messenger RNA (mRNA) transcript. Once found, one of the proteins in RISC, called ____2____, activates and cleaves the mRNA. This process is called RNA interference (RNAi) and it is found in many eukaryotes, and is a key process in gene ____3____ and defense against viral infections.
What is the difference between siRNA and miRNA?
How does this alter functionality?
siRNA has perfect complimentary to target RNA, whereas miRNA binding is imperfect.
This causes siRNA to lead to the degradation of the target RNA, whereas miRNA just leads to a block in translation.
- Both prevent translation, but in different manners
miRNAs and siRNA