Question

Development of an aptamer to bind the biomarker HER-2 (25 pts)

a) How will you create a starting aptamer library?

Draw an aptamer library member (RNA) and indicate the size of any constant or variable regions. Estimate how many aptamers would be in your starting library and give reason for this.

b) Draw the selection and enrichment cycle for isolating aptamers that bind HER-2. Be sure to label any primers, enzymes, or enzyme binding sites you need.

c) After two rounds of selection, there are about 100 unique aptamers still in your library. You wish to identify the strongest-binding aptamers from this enriched “pool” of aptamers.

What type of sequencing technique would you use, and why?

How could you use the sequencing data to determine which aptamer is the strongest binding?

d) An enriched aptamer pool can be diversified (mutated) to create a new library. What is the benefit of mutating the enriched pool to create a new library? How could you create a new library from your enriched pool of aptamers?

e) Your aptamer drug fails clinical trial because it binds other proteins in the blood serum (it is nonspecific).

How would you modify the Selection step above to solve this problem?

Answer 1

ANSWER A:-

• An aptamer is either a DNA/RNA sequence or protein which shows high binding affinity towards a molecule of interest such as Her-2 (Human Epidermal Growth Factor Receptor - 2) as mentioned in the query.
• To create a starting aptamer library, a random sequence of double stranded DNA is mostly preferred. This is the sequence where one is able to spot the constant region domain that provides specific towards the interaction. This region is used for the biological binding and can be amplified. So, a random sequence is generated as a starting point for selection.
• For this process, SELEX (Systematic evolution of ligands by Exponential enrichment) system is needed. Due to absence, I have referred to a monoclonal antibody called as Herceptin which shows the highest binding specificity and affinity to Her-2 protein.
• The sequence is as follows along with constant regions marked in bold at the terminals of the sequence.

GGCCA GGGCA CAAGG UGGAG AUCAA GCGUA GCCUG CUGCA CCAUC UGUCU UCAUC UUCCC GCCAU CUGAU GAGCA GUUGA AAUCU GGAAC UGCCU CUGUU GUGUG CCUGC UGAAU AACUU CUAUC CCAGA GAGGC CAAAG UACAG UGGAA GGUGG AUAAC GCCCU CCAAU CGGGU AACUC CCAGG AGAGU GUCAC AGAGC AGGAC AGCAA GGACA GCACC UACAG CCUCA GCAGC ACCCT GACGC UGAGC AAAGC AGACU ACGAG AAACA CAAAG CAGAC UACGA GAAAC ACAAA GUCUA CGCCU GCGAA GUCAC CCAUC AGGGC CUGAG CUCGC CCGTC ACAAA GAGCU UCAAC AGGGG AGAGU GUGAG CCAAA AUCCU GUGAC AAGAC UCACA CGUGU UGAGG AUCCC CCGAC CUGCG

• The above sequence indicates the RNA sequence of the antibody directed towards Her-2 protein. According to this, at least 1000 aptamers needs to be in the starting library. This has been the case in most of the RNA-associated aptamers and protein aptamers. But, to be specific, this depends mainly upon the abundance and the p-value for the given sequence of aptamer. Abundance indicates the frequency of the particular aptamer sequence in the entire pool of 1000 sequences and the p-value is calculated on the basis of enrichment cycles carried out. This earlier estimation helps to determine the number of sequences to be preferred. The p-value of all the sequences should be less than 0.01 and if this criteria is considered and is adhered to, then the number of aptamers to be used for screening can be optimized.

Note:- Due to unavailability of SELEX system, the antibody Herceptin has been characterized and evaluated for answer A. Any doubts or for more explanation, please prefer comment section.