IGEM:Harvard/2006/Adaptamers/Overview

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Background
An adaptamer is a nucleic acid that serves as a physical link or adapter between two substrates. Adaptamers have been made feasible through the discovery and development of aptamers, nucleic acid sequences that are capable of binding particular substrates with both high affinity and high specificity, i.e. the nucleic acid analogs of antibodies. Incorporation of two such aptamer sequences into a single nucleic acid complex gives adaptamers their adapter function.

The term adaptamer was coined by Dr. James and collaborators who designed the first adaptamer by directly fusing together two aptamers. In principle, this adaptamer should have been able to bind to two distinct protein targets. However, they found that the purported adaptamer was unable to bind at least one of its targets. In their seminal paper, Dr. James et al. attribute the problem to “inter-domain interactions” between aptamer sequences, preventing the adoption of conformations necessary for protein-binding. To get around this problem, these researchers created two separate oligonucleotides containing aptamer sequences. The first contained a protein-binding domain fused to CopA while the second aptamer contained a protein-binding domain of distinct specificity fused to CopT. The bulky secondary structure resulting from the specific interaction between CopA and CopT prevented interdomain interactions and thus led to the first functional adaptamer (see gallery).

While reliable, this system has some limitations. Due to their length (about 200 nucleotides) the chemical synthesis of the two components of the adaptamer becomes a challenge. Additionally, the bulk of CopA/CopT complex, targets are separated by a distance that precludes substrate interaction. We hypothesized that any small sequences forming a stable secondary structure between aptamer sequences would be sufficient for creating a functional adaptamer (see gallery). By using a short double-stranded region between aptamer sequences, it would become possible to quench adaptamer function following addition of a complementary displacement strand (see gallery).

Why Adaptamers?
Purely as a concept, adaptamers are interesting, theoretically providing a way to link any two substrates on a molecular scale. One can imagine using adaptamers to enhance protein-protein interactions, in effect creating a DNA or RNA (ribozyme) catalyst. Since aptamers can be evolved to bind to entire cell surfaces or even whole organisms, adaptamers could also link these types of substrates. Finally, adaptamers could be used to link two cell-types (possibly the same). This last goal would permit the study of the effect of increasing or stabilizing cell-cell interactions. While other strategies are possible for all these applications, adaptamers offer the advantages of being small and requiring no chemical modifications of substrates.

Results
Our main achievement this summer was the design of four adaptamer sequences that could link together streptavidin and thrombin. Our designs consisted of streptavidin and thrombin aptamers linked together by a variable length double-stranded linking region. Our first achievement was to create a program that could determine the sequence of the linking region between thrombin and streptavidin aptamers. This was necessary in order to prevent adoption of secondary structures that might inhibit thrombin or streptavidin binding (see gallery). We used this program to determine the sequences of our designs, then demonstrated that our adaptamer functions were functional using two assays. See gallery and 8/18, 8/25 for data. We also tested the ability to quench adaptamer function through the addition of a displacement strand that dissociates the two protein-binding regions of the adaptamer. Low concentrations of displacement strand had the desired effect of reducing the amount of thrombin targeted to streptavidin beads (see gallery). However, higher concentrations had the opposite effect and actually increased thrombin targeting to streptavidin beads (see 8/28 for data). We propose a lever hypothesis for this observation (see gallery).

Future Plans
We are currently testing an adaptamer that can target thrombin to a Jurkat cells. Additionally, we will test the lever-hypothesis proposed for the non-intuitive results seen in the strand displacement experiment (see 8/28).