Intellectual Merit: Aptamers are short single-stranded oligonucleotides. They are chemically produced, have the capability of binding with high affinity and specificity a variety of different targets, and have been shown to be useful as therapeutic agents and diagnostic tools. Their affinities are often comparable to those observed for monoclonal antibodies, and are significantly higher compared to those of peptides. As aptamer-amphiphiles, aptamers attached to a hydrophobic tail, they can assemble with other amphiphilic molecules into a variety of different structures. Their assembly with lipids to form aptamer-functionalized liposomes will be desirable in biotechnological applications, such as, targeted drug delivery. The PI proposes to study aptamers as aptamer-amphiphiles and, in particular, aptamer sequences that bind fractalkine (an adhesion molecule expressed only at sites of infection or inflammation, such as cancer), and hypothesizes that the orientation of the aptamer headgroup, and the use of a spacer between the hydrophobic tail and the headgroup, will affect the assembly behavior of the molecule, and the secondary structure of the aptamer, thus subsequently affecting its binding affinity for its target. To test this hypothesis the following research tasks have been set: In Research Task 1, the goal is to test the biological affinity of the designed aptamer-amphiphiles, free in solution, and incorporated in liposomes, thus evaluating which designs bind to fractalkine. The PI will characterize the aptamer-amphiphiles in Research Task 2 with different techniques (CD, melting curves, cryo-TEM, SAXS, and SANS) that will evaluate the secondary structure of the aptamer, and assembly behavior of the amphiphiles. These results will enable a connection between the biological function of aptamer-amphiphiles with their structure and corresponding phase behavior. This research will advance knowledge and understanding as it will not only furnish favorable amphiphilic properties (headgroup orientation, spacer design, secondary structure, assembly behavior) for the design of aptamer-amphiphiles, but will provide a fundamental understanding of why these properties are beneficial.

Broader Impact: -Technical Impact. The PI proposes the design and characterization of a novel tool, "aptamer-amphiphiles", based on the expectation that by changing their structural segments one can control their morphology, surface chemistry, and function. A comprehensive characterization of secondary structure, amphiphilic and self-assembly properties, and the link between these properties and the engineering of targeted delivery vehicles is proposed here for the first time. The research activities represent transformative research as they may lead to the genesis of a new field of research on "aptamer-amphiphiles" (a new term in the literature) that will merge rational amphiphile design principles, chemistry, biology, and engineering in order to provide advanced systems for targeted drug delivery.

-Societal / Educational Impact. Fractalkine is an attractive new target for the development of anti-angiogenesis and anti-cancer agents. By choosing aptamers that bind to fractalkine as the model system, the proposed work outlines a logical approach that can be applied to the future design of aptamer-amphiphiles that can functionalize nanoparticles and increase the particle's specificity and affinity for fractalkine, thus having great therapeutic potential and significantly benefiting society. The educational plan is strongly integrated with the research plan. The PI is collaborating with "DragonflyTV" (a science series that presents middle school students doing real science, supported by NSF and produced by Twin Cities Public Television (TPT), and seen on Public Broadcasting Service (PBS) stations nationwide) with the goal of enhancing scientific and technological understanding among children and adults. Furthermore, the PI has participated, and continue to participate, in outreach activities organized by TPT with the goal of attracting women in sciences and engineering (a long term goal for the PI), and is currently collaborating, and will continue to do so in the future, with MRSEC. Through MRSEC the PI will be hosting during the summer high school teachers, and minority undergraduates from a nationwide pool of applicants, through a well established network of high schools, and minority undergraduates from a number of Minority Serving Institutions.

Project Report

The hypothesis of this work was that when designing aptamer-amphiphiles the orientation of the aptamer headgroup and the use of a spacer between the hydrophobic tail and the aptamer headgroup will affect the binding affinity of the aptamer for its target, the secondary structure of the aptamer and the assembly behavior of the amphiphile. Aptamers are ssDNA oligonucleo­tides that can naturally fold into different 3D structures in a variety of environments. They have the capability of binding with high affinity a variety of different targets and rival antibodies in terms of affinity and specificity for their targets. The focus was on aptamers that specifically targeted fractalkine, an adhesion molecule on the surface of inflamed endothelial cells and certain cancer cells, such as prostate, lung, and colorectal cancer. Multiple runs of SELEX (Systematic Evolution of Ligands by Exponential Enrichment) were necessary to identify an aptamer, FKN-S2, that bound to fractalkine with high affinity and specificity. Synthetic routes were identified that allowed for the synthesis of aptamer-amphiphiles while varying the type and length of the spacer used between the aptamer and the hydrophobic tail of the amphiphile. The FKN-S2 aptamer headgroup was either conjugated directly to the hydrophobic tail (C16 double tail) with no spacer (NoSPR) in between, or with polyethylene glycol (PEG4, PEG8, PEG24), alkyl (C12 and C24), or oligonucleotide (T10 and T5: 10 and 5 thymine, and A10: 10 adenine) spacers at its 3’ or 5’ end to create different FKN-S2 aptamer-amphiphiles. The orientation of the aptamer and the presence of a spacer was found to have a pronounced effect on the binding affinity of the FKN-S2 aptamer-amphiphile for fractalkine. This was the first systematic study to show the effect of a spacer on the binding affinity of an aptamer-amphiphile for its target. Circular dichroism (CD) spectroscopy and thermal melting studies indicated the aptamer forms a stem-loop and intramolecular parallel G-quadruplex , and the tail strongly stabilized the formation of the G-quadruplex in a buffer. Parallel G-quadruplex structures are tertiary DNA structures formed by the stacking of G-quartet structures, with each G-quartet formed by four guanine (G) nucleotides arranged in a planar, square geometry held together by Hoogsteen hydrogen bonding. Cryogenic transmission electron microscopy (cryo-TEM) imaging showed the aptamer-amphiphiles self-assembled into micelles and nanotapes, flat bilayer structures that were often twisted. The study was extended to include other aptamers such as the Muc-1 aptamer that binds to the Muc-1 glycoprotein. Cryo-TEM revealed the existence of weakly ellipsoidal globular micelles within the NoSPR, PEG4 and PEG8 spacer amphiphiles. However, the C12 and C24 spacer amphiphiles, in addition to forming micelles, also self-assembled into bilayer nanotape structures that were flat or twisted. Data from small angle x-ray scattering (SAXS) experiments supported the existence of multiple morphologies of the aggregates seen in the cryo-TEM images. The unmodified Muc-1 aptamer (free aptamer) had a CD spectrum that was characteristic of stem-loop structure. Similar spectra were seen from Muc-1 aptamer-amphiphiles with PEG4 and PEG8 spacers, which suggested that the assembly process did not significantly alter the secondary structure of the aptamer headgroups. In contrast, the C12 and C24 spacer amphiphile spectra closely matched the expected CD spectrum of parallel G-quadruplexes. The effect of the ssDNA length and secondary structure on the self-assembly behavior of the ssDNA-amphiphiles was also investigated by designing amphiphiles in the presence and absence of the polycarbon spacer. It was found that bilayer nanotapes were formed that transitioned from twisted nanotapes, to helical nanotapes to nanotubes. The DNA nanotubes were formed using a variety of headgroup lengths and sequences. This was the first time that DNA nanotapes and nanotubes were observed from the self-assembly of ssDNA-amphiphiles.

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University of Minnesota Twin Cities
United States
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