2015 Participants
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NAME: Tyler Skidmore

Effect of Doxorubicin, Fe2+, and Mn2+ on the binding between IRP and IRE

The goal of the previous project was to synthesize high-stability DNA conjugates in order to form self-assembled monolayers (SAMs).  Such conjugates have many applications in the biosciences – such as genetics and agriculture – and allow much room for further research.  These conjugates can function in the same way as a protein biochip and can capture analytes from solution.  The DNA conjugates consist of a cleaved cystamine-core polyamidoamine (PAMAM) dendrimer (dendron) as a headgroup, a NHS-Maleimide linker, and an extending DNA sequence as shown in Figure 1.  The experimental design was to change different parts of the conjugate in order to improve the stability of the resulting SAM.

Specifically, I worked with Destiny Carte and Chris Warner to primarily study the effect of the linker on SAM stability.  My contributions to the project included synthesis and optimization of the conjugates as well as data gathering with SPR.  Concerning the experimental procedure, generation 4 PAMAM dendrimers were used as headgroups in the conjugates.  The linkers sSMCC, GMBS, and AMAS were used to connect the headgroup to the capturing DNA.  Agarose gel electrophoresis and extraction was used to purify and quantify the extent of the DNA-dendron conjugate reaction.  Following extraction with TBE, the concentration of the resulting conjugate solution was determined with a Nanodrop.  The conjugates were allowed to self-assemble onto a gold surface and SPR reflectometry was used to quantify the binding efficiency of the capturing DNA with the analyte (target DNA).

Following analysis of the data generated from SPR reflectometry, it was determined that sSMCC functioned as the most efficient linker.  Compared to the other linkers that were tested, sSMCC possesses an intermediate length and structural hindrance.  This previous work provides a solid base for the IRP/IRE study.  In the research concerning the IRP/IRE complex, a generation 4 PAMAM dendrimer with the linker sSMCC will be used to form a SAM on a gold surface which will interact with the IRE mRNA.