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Abstract:

Title: "Structural and biochemical evaluation of natural and modified Nucleic Acids and their interactions with drugs and biomolecules for th erapeutic and synthetic biology application" Deoxyribo and ribo nucleic acids (DNA and RNA) are the two key players i nvolved in the ‘central dogma’ of molecular biology (DNA to RNA&nbs p;to Protein). Generally, DNA acts as the storage of genetic information while RNA molecules for long time are being considered as the intermedi ates to transfer the genetic information from DNA to the functional prot ein world. However, this notion regarding RNA is significantly altered b y some of the recent decades’ discoveries, where RNAs are shown to have catalytic function (by ribozymes) and a wide regulatory role in gene-exp ression by endogenous non-coding RNAs like microRNA (miRNA) and by short interfering RNA (siRNA) mediated RNA interference (RNAi). This in turn has provided us the novel opportunities to target functionally deregulat ed RNAs involved in pathogenesis. Targeting the RNA level in some diseas es can be advantageous over protein targets where there is a lack of str ucturally drugable proteins. On the other hand, targeting at the DNA lev el is difficult since nuclear membrane and highly packed DNA provide a b arrier for the access to the drug molecules. Targeting in the RNA level can be achieved either by small molecular approach or by using modified oligonucleotides (e.g. antisense agent, modified-siRNA/miRNA etc). Each approach has its inherent merits and limitations as well.Natural RNA adopts diverse secondary and tertiary structures, and possesses necessa ry structural flexibility that is crucially important for its biological function. Structural understanding of the RNA and its interactions with other RNAs, biomolecules and small molecule ligands is necessary for th e understanding of their biological function and for the design of suita ble drugs that target RNA. In the case of RNA targeting by modified olig onucleotides, they should mimic the natural recognition principles and t herefore their three-dimensional structure has to be compatible with the existing natural RNA and protein machinery. Beside the therapeutic appl ication of chemically modified oligonucleotides, recent studies demonstr ate a growing interest in chemically modified nucleic acids for their po ssible application in synthetic biology. These oligonucleotides should b e structurally and biochemically orthogonal to the natural nucleic acids but should be able to carry out storage and transfer of genetic informa tion independently inside a living cell. Here as well the structural and biophysical studies of chemically engineered alternative information sy stems and their comparison with the natural DNA/RNA are necessary for th e selection of suitable candidates for further biological evaluation. Th e comparative study can also answer the questions related to ‘why nature has selected DNA/RNA as the genetic information systems’ rather than an y other nucleic acids.The second chapter (first project) of this the sis describes the identification and characterization of secondary struc tural forms of mature miRNAs. miRNAs are a class of endogenous, short, non-coding RNAs that post-transcriptionally regulate gene-expression by b inding with partially complementary sequences in mRNA and thereby steric ally blocking translation or by triggering mRNA degradation. An equilibr ium between hairpin and homo-duplex form of the hsa-mir-520h mature miRN A strand has been characterized by using solution state 1D and 2D proton NMR spectroscopy. A detailed sequence analysis was performed for all mi RNAs from the database of known miRNAs (miRBase). The statistical analys is of the resulting folding and alignment data revealed the potential of a large number of miRNAs to form significant self-complementary hairpin and/or homo-duplex structures in solution under physiological condition s. Several mechanistic models are discussed by which self-complementarit y of mature miRNAs can provide a mechanistic tuning and a regulatory sop histication to the process of miRNA mediated gene regulation.The thi rd chapter deals with RNA-targeting by small-molecular approach for a ne wly emerging therapeutic RNA target, pre-microRNA. Over-expressions of m iRNAs are being increasingly linked with many diseases including differe nt types of cancer. In this study, the role of some known small molecula r therapeutics has been investigated for their ability to bind with a pr e-miRNA (hsa-mir-155) and thereby to interfere with the final step of mi RNA biogenesis, in which an RNase III enzyme Dicer catalyzes the formati on of mature miRNA from the corresponding inactive stem-loop hairpin (pr e-miRNA) substrate. Potential binding and inhibition effects have been d emonstrated by some of these analogues. They can be used as leads for fu rther development of potent and selective small molecular miRNA-antagoni sts.In the fourth chapter structure determination of several altrito l nucleic acids (ANA)-modified siRNAs was performed and binding of sever al ANA and thymidine residue (dTdT)-modified siRNAs were investigated wi th the PAZ domain of Argonaute 2 protein of Drosophila melanogaster. ANA contains six-membered altritol sugar in the sugar-phosphate backbone. A NA-modified siRNA (ANA/RNA) duplexes adopt a geometry very similar to th e naturally occurring A-type siRNA duplex. The six-membered altritol sug ar in ANA occurs in a chair conformation with the nucleobase in an axial position. Binding studies demonstrate that modifications in the double- stranded region of the antisense strand have some small effects on the b inding affinity. Modification of the 3′ overhang (unpaired protrud ing part of a DNA/RNA duplex) with dTdT shows a six-fold increase in the binding affinity compared with the unmodified siRNA, whereas modificati on of the 3′ overhang with ANA largely decreases the binding affin ity. The structural and binding data of this study in combination with t he previous results of biological (RNAi) activity shed light on the unde rstanding of the therapeutic potential of chemically modified siRNAs (as RNA-targeting tools) from a structure-function point of view, and highl ight for the future therapeutic design of chimeric modified siRNAs with improved protein recognition and RNAi activity. The fifth chapter describes the structural and biochemical evaluation of de oxyxylonucleic acid (dXNA) as an orthogonal genetic information system f or synthetic biology application. Solution structure and conformational analysis of two self-complementary, fully modified dXNA oligonucleotides are presented, as determined by CD and NMR spectroscopy. These studies are the initial experimental proof of the structural orthogonality of dX NAs. In aqueous solution, dXNA duplexes predominantly form a linear ladd er-like (type-1) structure, which is significantly different from natura l DNA/RNA duplex structure. The corresponding nucleoside triphosphates ( dXNTPs) were synthesized and evaluated for their ability to be incorpora ted into a growing DNA chain by using several natural and mutant DNA pol ymerases. Despite the structural orthogonality of dXNA, DNA polymerase&n bsp;ß mutant is able to incorporate the dXNTPs, showing initial DNA -dependent dXNA polymerase activity.