Cells are primarily comprised of metal ions, small molecules, proteins, lipids, and nucleic acids. The ability to probe these molecules in single living cells can shed new light on chemical processes inside of cells or allow disease diagnosis based on molecular profiling. However, there exists a lack of tools that allow one to monitor and analyze these molecules dynamically in live cells. Although genetically-encoded fluorescent tags have transformed live-cell protein analysis, there is a deficiency of robust techniques for studying other molecules. In this regard, probes based on nucleic acids have recently emerged as powerful tools for studying intracellular processes. Their biocompatibility, amenability to genetic encoding, low cost, ease of synthesis, modular structure, and ability to be chemically modified in a sequence-defined manner make them especially useful in sensing applications. By tuning their sequence, nucleic acids can be designed to recognize a wide range of molecules including other nucleic acids, proteins, ions, and small molecules. The earliest analysis techniques based on DNA, such as in situ hybridization and polymerase chain reaction (PCR), required the fixation and lysis of cells, respectively, preventing their use for live-cell analysis. The introduction of linear DNA probes that can study events in live cells, such as molecular beacons, helped to expand the capabilities in the field. However, such DNA probes do not efficiently cross the cell membrane without the use of transfection reagents, and they are susceptible to rapid nuclease degradation in the cellular environment. To overcome these challenges, NanoFlares were developed in 2007 as a new tool for live-cell analysis. NanoFlares are comprised of a gold core functionalized with recognition strands (hybridization-based, aptamer, DNAzyme, or aptazyme) for a target of interest. These recognition strands are hybridized to short fluorophore-labeled flare strands. Close proximity between the gold nanoparticle and the fluorophore quenches the fluorescence. When the target is present and binds to the recognition strand, the flare strand is displaced, separating the fluorophore and gold, and turning on fluorescence. Owing to the dense orientation of DNA on the nanoparticle surface in a spherical nucleic acid (SNA) architecture, NanoFlares exhibit high cellular uptake without the need for transfection reagents, display enhanced resistance to nuclease degradation in comparison to free nucleic acid probes, have enhanced target recognition and binding, and exhibit little immunogenicity or toxicity. To date, NanoFlares have been used in over 50 studies for studying various targets including mRNA, small molecules, ions, and proteins. Although NanoFlares constituted the first platform for live intracellular analysis at single-cell resolution, challenges still exist. These challenges include false-positive signal due to non-specific separation of the fluorophore and gold nanoparticle quencher, limited quantitative capabilities, inability to spatiotemporally track analytes, kinetically slow responses due to partial blocking of the recognition strand, and the restriction that only targets with known nucleic acid-based recognition sequences can be detected. My dissertation research seeks to alleviate these challenges through the development of next generation SNA constructs for live cell chemical analysis. In Chapter 2, a new class of quencher free signaling aptamers called Forced-intercalation aptamers (FIT-aptamers) are introduced. It is shown that FIT-aptamers offer several advantages over state-of-the-art transduction methods, and enable study of important analytes such as markers of cancer, thrombosis, and heavy metal poisoning in complex media. Chapter 3 explores the leveraging of these advantageous properties to design the first fluorogenic aptamers capable of sensing steroid hormones in clinical serum samples. Chapter 4 reports the development of a new class of live cell probes based on protein spherical nucleic acids (ProSNAs). ProSNAs are able to recognize analytes using either DNA-based or protein-based recognition, ultimately enabling false-positive resistant measurement of analytes in living cells. Finally, Chapter 5 discusses the outlook and future directions for the work covered in this thesis.

Creator

• Ebrahimi, Sasha

DOI

• https://doi.org/10.21985/n2-5zvs-ap26

Subject

• Chemical engineering

Language

• en

Alternate Identifier

• etdadmin_upload_829912
• http://dissertations.umi.com/northwestern:15624

Date created

• 2021-01-01

Resource type

• Dissertation

Rights statement

• In Copyright