Throughout the iGEM 2021 season, our team has made many significant discoveries and products that may prove useful for future iGEM teams. Our findings are presented as follows.
After reviewing literature, we have evaluated the 3D structure (as well as the full amino acid sequence) of human sclerostin.
Sclerostin has three loops present in its secondary structure[1], illustrated below in Fig E.1:
Fig E.1: 3D NMR structure of sclerostin[1].
The sclerostin protein, with a length of 213 residues, has a secondary structure that has been determined by protein NMR to be 28% beta sheet (6 strands; 32 residues)[2]. It is produced primarily by the osteocyte but is also expressed in other tissues,[3] and has anti-anabolic effects on bone formation.[4] Mutations in the gene (SOST) that encodes the sclerostin protein are associated with disorders associated with high bone mass, sclerosteosis and van Buchem disease[5].
After reviewing literature, we have determined that our project will use aptamers over antibodies to combat against the low bone density caused by osteogenesis imperfecta.
In order to have both high selectivity and high affinities, the agent used should recognize a broad surface area on the protein. Generally, small molecules do not have sufficient surface area to bind to a large enough surface of the protein to generate nanomolar (nM) binding affinities that can differentiate local modifications. Traditionally, these requirements have been met by antibodies, developed as the affinity ligands of choice for identifying proteins in research, diagnostics, biosensors, imaging, and therapeutics[6].
Although monoclonal antibodies (mAbs) provide highly selective, high binding affinity ligands, they have significant limitations[7]. Selection difficulties, selectivity problems, preparation difficulties, high costs of production, stability and cross reactivity issues are the major limitations in using monoclonal antibodies as detection reagents.
Not only that, in most proteomic investigations, mass spectrometry (MS) is used as the analytical method of choice to quantitate the target protein. In the majority of cases, particularly in the case involving protein biomarkers (e.g. involving inflammatory cytokines, relevant to our project), the target protein would be very low in concentration relative to that of the antibody[6]. The abundant signals from the antibody will also interfere with the MS signal detection signals of the protein of interest, particularly after the digestion of antigen/antibody complexes[6].
On the other hand, not only are aptamers 1/10th of the molecular weight of antibodies, they have been shown to mimic antibodies and exhibit high binding affinity, having dissociation constants typically in the nano-molar and even pico-molar range, and high selectivity towards their targets[8-12]. The detection limit of zeptomole (10-21 mol) amounts was achieved with DNA aptamers[13].
This is because the ability of single stranded nucleic acids (ssNA) to fold into unique and stable secondary structures allow aptamers to form tertiary structures that not only recognize and bind specifically to protein targets, but also discriminate between subtle molecular differences within the target [14-16].
There are numerous advantages of aptamers over antibodies[17]:
Combining all these reasons, we have determined that aptamers are superior to antibodies for our project.
Details about the process proper can be found here.
To track the progress of a SELEX reaction, the number of target bound molecules, which is equivalent to the number of oligonucleotides eluted, can be compared to the estimated total input of oligonucleotides following elution at each round[21].
A typical aptamer selection process via traditional SELEX can range from weeks to months, and tales 10-20 SELEX cycles to complete. This presents a temporal and quality restriction and impedes aptamer development and application, especially in therapeutic contexts[22]. As such, our team has incorporated two different SELEX protocol modifications to speed up the aptamer production process:
Counter SELEX[23]:
During the selection period, some of the sequences might unwantedly bind to the immobilization matrix, causing false positive results. To address this issue, an additional step is added: using structurally-similar targets to incubate with aptamers to discriminate non-specific oligonucleotides. This vastly increases the affinity of aptamers in the final yield of SELEX.
Microfluidic SELEX[24]:
The microfluidic system includes reagent-loaded micro-lines, a pressurized reagent reservoir manifold, a PCR thermocycler and actuatable valves for selection and sample routing. This vastly increases the speed of the SELEX process by reducing the amount of cycles needed to achieve a certain high affinity.
Further details can be found here.
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