MSE Ph.D. Defense – Maeling Tapp

Date: Friday, December 19, 2014


Location: MoSE, room 4202

Time: 1:00 pm

Committee members:


Dr. Valeria Milam (MSE, Advisor)

Dr. Kenneth H. Sandhage (MSE)

Dr. Christopher Summers (MSE)

Dr. Vladimir Tsukruk (MSE)

Dr. Philip Santangelo (BME)

Dr. Rajesh Naik (AFRL)

Title: Competition-Induced Selection of Ligands for the Screening of ssDNA Aptamers for Gold Substrates

Abstract:

Aptamers are single-stranded oligonucleotide sequences that exhibit high affinity and high specificity binding for nonnucleotide targets. Using in vitro selection processes, aptamers have been identified from combinatorial libraries consisting of ~ 1012-1015random sequences for a variety of targets including but not limited to ions, small macromolecules, and whole cells. The most commonly used screening technique for identifying aptamers is widely known as Systematic Evolution of Ligands by EXponential Enrichment (SELEX) which is an iterative process with each selection round consisting of three main stages: incubation of random oligonucleotide library with target, partitioning and finally elution and amplification of the bound aptamers.

Due to their small molecular weight, ease of processing, and long-term stability, aptamers are now increasingly explored as potential alternatives to antibodies as high affinity ligands. These characteristics highlight the potential impact of aptamers in areas such as biosensing, diagnostics, and therapeutics. While aptamers have been identified for a number of biologically-based targets, few studies have investigated nonbiological materials as targets. This thesis presents gold nanoparticles (AuNPs) as a target of interest due to their unique optoelectronic properties that are size- and shape-dependent and thus provide advantages for use as biological sensors and in therapeutic and medical diagnostic applications. Specifically, interactions between oligonucleotides and AuNPs have become a great area of interest, as DNA-functionalized AuNPs have provided even greater flexibility for their use in the development of controlled nanoassemblies, biodiagnostics and cellular imaging.

Part I of this thesis presents an investigation into the effect of nucleic acid additions on the seed-mediated growth of AuNPs and reveals base-specific and structure-specific effects through the use of spectroscopic analysis. Part II focuses on the development of an alternative aptamer screening process, the competition-induced selection of ligands (CISL) for the identification of aptamers for gold substrates. The CISL approach differs from conventional SELEX-based approaches in that it (i) shortens the selection process, (ii) promotes increased competition during selection rounds and (iii) reduces PCR amplification-induced artifacts in the candidate sequence pool. Part III details the use of the CISL approach for the screening of aptamer candidates for AuNPs and planar gold substrates. Identified aptamer candidates were then systematically compared using various analytical tools to reveal similarities in their primary sequence structures and their predicted secondary structures to probe the nature of the aptamer-target binding.