Exploring alternative bioconjugation methods for covalent linkage of DNA and peptides
Abstract
The field of bioconjugation focuses on creating combinations of biological and non-biological molecules for novel or increased functionality. Although this encompasses a myriad of chemical structures, we are primarily interested in the conjugation of oligonucleotides and peptides. Oligonucleotide-peptide bioconjugate applications include biological assays, cellular delivery, protein immobilization, and specialized electrophoresis techniques. Current bioconjugate techniques are tedious, expensive, or introduce undesired chemical groups. In order to increase the feasibility of current applications and to open new avenues for bioconjugates, we have attempted to develop two new bioconjugation techniques, one using L-DOPA crosslinking chemistry and one using EDC crosslinking chemistry. Using RP-HPLC, UV/vis spectrometry, MALDI mass spectrometry, and 2D ESI mass spectrometry, we have explored the efficacy of both methods. Under the reaction conditions employed, periodate-mediated DOPA crosslinking chemistry is not an appropriate method for covalently linking DNA and peptides, although the DOPA peptide does undergo changes upon oxidation; no conjugates were formed. Using a two-step reaction with intermediate purification, EDC was shown to be effective in creating conjugates between phosphorylated DNA and neutral or positively charged peptides, but it was unable to form a conjugate between phosphorylated DNA and a negatively charged peptide. This opens the door for further bioconjugation research using EDC crosslinking chemistry.
Degree
M.S.E.
Advisors
Tao, Purdue University.
Subject Area
Biochemistry|Biomedical engineering
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