Characterization [of] nucleic acids unwinding and exploring its application in miRNA detection in the nanopore

Title from PDF of title page (University of Missouri--Columbia, viewed on May 15, 2013).The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file.Dissertation advisor: Dr. Li-Qun (Andrew) GuIncludes bibliographical references.Vita.Ph. D. University of Missouri--Columbia 2012."May 2012"MicroRNAs (miRNAs) are a class of short (~14-27 nucleotides) noncoding RNAs that regulate gene expression at the post-transcriptional level. Aberrant expression of miRNAs has been found in all types of tumors. Reverse transcription real-time polymerase chain reaction (qRT-PCR) and microarrays have been developed for detecting microRNA; however, these methods need labeling and amplification, and suffer from cross-hybridization, low selectivity and lack of valid internal controls. The development of nanopore sensors for microRNA detection is a new effort. One of the superior properties of nanopore is that the ion current in a nanometer-scaled pore structure is very sensitive to the presence, location and conformation of single target molecules occupying the ion pathway. Here we show that a nanopore sensor based on the alpha-hemolysin protein can selectively detect microRNAs at the single molecular level in the plasma samples of lung cancer patients without labeling and amplification of microRNAs. First, we uncovered a signature current pattern that can be used to electrically track the double-stranded DNA (dsDNA) unzipping process. With the signature signals, we can also distinguish the release of dsDNA without unzipping. Second, based on the electrical signatures we identified, we have designed a nanopore-based microRNA sensor for the direct and label-free detection of target microRNA in a fluctuating background, such as plasma RNA extracts from clinical samples. This sensor can quantify picomolar levels of cancer-associated microRNAs and can distinguish single-nucleotide differences between microRNA family members. This nanopore method can be a useful tool for quantitative studies of microRNAs, which are important for non-invasive screening and the early diagnosis of diseases such as cancer.Includes bibliographical reference