Access Type

Open Access Dissertation

Date of Award

January 2015

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Chemistry

First Advisor

Christine S. Chow

Abstract

Outbreaks of advanced antibiotic-resistant strains of microbes have hastened the need to identify new viable molecular targets for the development of novel anti-infectives. For this purpose, helix 69 (H69, or m3a 19-nucleotide (nt) hairpin motif that is highly conserved throughout phylogeny and rich in modified nucleotides, including pseudouridine () and 3-methylpseudouridine (m3) was chosen as a potential target. Helix 69, which is located in domain IV of Escherichia coli 23S ribosomal RNA (rRNA), undergoes conformational changes when in close proximity to the decoding region of 16S rRNA and transfer RNAs (tRNAs) in the peptidyl-transferase center (PTC). Functionally, the exact biological role(s) of H69 remains unclear; however, its proposed importance within protein synthesis may support it as an ideal target to develop ligands with high binding specificity.

In this thesis work, DNA aptamer candidates with binding specificity for the wild-type bacterial H69 were selected. The 84-nt-long DNA aptamer (H69DNAapt18) that was identified from a DNA library with a 40-nt randomized region has the sequence 5'-CTCCCCGGGCACTATTTCCTGGGACTAGTTCTGCAGGTTT-3'. The initial library contained 5 × 1014 DNA sequences and was used in SELEX (systematic evolution of ligands by exponential enrichment) (Molecular diversity of the library was reduced to approximately 1  102 after 11 rounds of in vitro selection) experiments. A synthetic construct of H69 was biotinylated and used with optimized SELEX. After immobilization of the biotinylated target H69 to streptavidin-coated surfaces, DNA library candidates were challenged against H69 in multiple rounds of selection, recovered, and enriched by direct-bead PCR (polymerase chain reaction). Levels of bound DNA and diversity of the amplified library pools were monitored by UV-visible spectroscopy and sequencing between rounds of SELEX (11 total). Select rounds were cloned and sequenced. Consensus sequences from select rounds of SELEX were identified by using Clustal W alignments, and optimal secondary structures were predicted by Mfold analysis. Analysis of 120 clones led to the identification of 20 sequences with consensus motifs. Notably, one of the selected DNA ligands (H69DNAapt18) contained a conserved 20-nt hairpin-loop motif with complementarity to the loop region of the targeted E. coli wild-type H69. Interestingly, this 20-nt hairpin motif of H69DNAapt18 retained its conserved 20-nt motif within the truncated 40-nt Mfold structural prediction representing only the randomized region of the 84-nt DNA library.

Attempts to determine binding affinities of select isolated DNA aptamer candidates to 32P-radiolabeled H69 by electrophoretic mobility shift assays (EMSAs) were moderately successful, with observation of RNA-DNA complexes with apparent dissociation constants (Kds) in the high nM range. To better evaluate the affinity and selectivity of DNA aptamer #18, a fluorescently tagged H69-DNA aptamer #18 (FAM-H69DNAapt18) was used for a comparison binding study with wild-type H69 and unmodified rRNA constructs. Relative dissociation constants of FAM-H69DNAapt18 for H69 and RNAs other than H69, as determined by fluorescence titrations with small RNA constructs, are as follows: wild-type H69 > partially modified H69 > unmodified H69 > A-site RNA of 16S rRNA.

Overall, this study provides a reference point for the development of DNA aptamers that identify modified nucleotides and/or methylation sites in RNA, or could potentially function as novel therapeutics to help combat antibiotic resistance.

Keywords: 23S ribosomal RNA (rRNA); helix 69 (H69); modified nucleotides; pseudouridine (); 3-methylpseudouridine (m3); peptidyl-transferase center (PTC); systematic evolution of ligands by exponential enrichment (SELEX); aptamers; fluorescence spectroscopy; DNA:RNA complexes; dissociation constant (Kd); anti-infectives; antibiotic resistance; antibacterial therapeutics; and bioprobes.

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