Methods For Detection Of MicroRNA And Impact Of Oxidative Lesions On RNA G?quadruplex

Non-coding RNA affects the expression of genetic information through indirect manner.This thesis focuses on two important non-coding RNA:microRNA and telomeric RNA.MicroRNA(miRNA)is a recently discovered non-coding,endogenous RNA that exists in eukaryotes,and usually consisting of only 19-23 nucleotides.A variety of regulatory functions of miRNA has been demonstrated.Through participating in RNA-induced silencing complex,miRNA could inhibit translation but not affecting the integrity of m RNA.Aberrant miRNA expression represents a common characteristic phenomenon in diseases,even carcinoma.Rapid and accurate quantification of miRNA is conducive to diagnosis of cancer development and prognosis,thus making miRNA an important biomarker in clinical diagnose and treatment fields.Telomeric RNA is transcribed by RNA polymerase through telomeric DNA,the length of the span is about0.1-9 kilobases.It turns out that telomeric RNA is existed in yeast cell,zebra fish,mice and human,the evolutionary conservation indicates the important research value of telomeric RNA.Although the intercellular functions of telomeric RNA have not been thoroughly studied,some biological functions of telomeric RNA have been discovered recently.For example,telomeric RNA might be involved in the regulation of chromosome recombination and the regulation of telomerase activity through the formation of DNA/RNA double strand or G-quadruplex.The oxidative damage product8-oxoguanosine is the main type of oxidative damage in RNA,and this damage is more likely to occur in guanine rich sequences.The N7 position of 8-oxoguanosine will affect the hydrogen bond which form G-tetrad.The study on the oxidative damage of 8-oxoguanosine provides an important guiding significance for the future study on the biological function of telomeric RNA.One of main research of this thesis focuses on building a DNAzyme logic gate to detect multiple miRNAs simultaneously.Due to its simple structure and high specificity,DNAzyme was adopted for amplified detection of miRNAs and constructing a series of DNAzyme logic gates.An additional duplex specific nuclease(DSN)was introduced into the constructed DNAzyme system to further amplify the readout signal by releasing the intact miRNA.Through the proper DNAzyme-grafting and an efficient DSN amplification,a series of YES,NOT,AND and INHIBIT logic gates were rationally designed and realized as highly accurate sensing platforms for simultaneously amplified detection of multiple low-abundance miRNAs(with a detection limit of 100f M)that correspond to a specific aberrant miRNA expression pattern of various diseases.This DSN-DNAzyme cascade system could discriminate carcinoma tissues from its corresponding para-carcinoma tissues,that we believe can extend the potential applications of DNAzyme-based computing circuits in bioanalytical and biomedical research.The second research of this thesis is to study the influence of 8-oxoguanosine on the structure and function of telomeric RNA by circular dichroism spectrum,T_m value determination,polymerase elongation and nuclear magnetic resonance spectroscopy.The influence of 8-oxoguanosine damage at each site on the structure of telomeric RNA G-quadruplex was studied by CD.Though T_m value determination,we studied the influence of oxidative damage to stability of G-quadruplex.The mismatch ability of 8-oxoguanosine was verified through the experiment of multiple polymerase elongation.Through two kind of polymerases applied in wild-type telomeric RNA sequence and modified 8-oxoguanosine telomeric RNA sequence to study extended stop phenomenon.At last,we used NMR analysis to study the structure of RNA G-quadruplex.