Part I: Design, synthesis and applications of oligonucleotides modified by lipophilic subsituents. Part II: Functional modification of pRNA

Brian M Laing, Purdue University

Abstract

Part I: The properties of oligonucleotides modified with lipophilic substituents were investigated by using a series of short, self-complementary oligonucleotide sequences modified at their 5'- and/or 3'-termini with a C12 hydrocarbon. These systems serve as models to assess the biophysical properties of double-stranded DNA (dsDNA) equipped with potentially stabilizing lipophilic substituents. Addition of C12 to the 5'-termini of self-complementary 10 nucleotide sequences increased their duplex melting temperatures (Tm) by ∼ 4–8 °C over their corresponding unmodified sequences. C12 functionalities added to both the 3'- and 5'-termini increased T m values by ∼ 10–12 °C. In addition, cellular uptake was significantly enhanced in mouse fibroblast cells, chicken B cells and human nasopharyngeal cancer cells. Stable, short dsDNA sequences are of potential interest in the development of transcription factor decoy oligonucleotides as possible therapeutic agents and biological tools. These results suggest that the stability and cellular uptake properties of short dsDNA sequences are improved by strategic placement of lipophilic substituents and can be used as the basis for the design of dsDNAs with improved biological stabilities and function under physiological conditions. Part II: The ability of packaging RNA (pRNA) from the phi29 DNA packaging motor to form multimeric nanostructures by loop/loop interactions, has been exploited for the development of the nascent field of RNA nanotechnology and subsequent applications in nanomedicine. For applications in nanomedicine, it is necessary to modify the pRNA structure for the conjugation of active molecules. We investigated two approaches for the modification of pRNA. In the first approach, we have developed an optimized synthetic strategy for linking cellular targeting molecules to 5'-AMP, which can then be used to label the 5'-end of pRNA by T7 RNA polymerase mediated synthesis. By using a boronate affinity gel for purification, we have developed an efficient route to folate conjugated AMP with high yields and purity. For the second approach, we have investigated capped double-stranded DNA segments as reversible capture reagents for pRNA. The results of model studies show that 5- to 7-nt overhangs on a target RNA can provide efficient handles for the high affinity association to capped double-stranded DNA.

Degree

Ph.D.

Advisors

Bergstrom, Purdue University.

Subject Area

Biochemistry|Organic chemistry

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