Exploring Partially Morpholino-Modified siRNAs and their Interactions with the Argonaute-2 Protein

Hage, Jeffrey R

Undergraduate Honors Thesis

Exploring Partially Morpholino-Modified siRNAs and their Interactions with the Argonaute-2 Protein Pubblico Deposited

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Citeable URL: https://scholar.colorado.edu/concern/undergraduate_honors_theses/cc08hh12q

Abstract

Biochemists are fascinated with protein levels. An incredible amount of time has gone into researching how relative levels of specific proteins correlate with cancer. With the discoveries found by these scientists, a vast field of therapeutic potential lies in wait on the ability to attenuate levels of specific proteins in humans. Researchers have been exploring the usage of siRNA, a type of biomolecule that can decrease specific target protein levels in-vitro, as a possible in-vivo treatment. However, siRNAs struggle in animal systems due to a variety of barriers such as membrane permeability and degradation. In an attempt to overcome this, researchers have developed a wide variety of chemical modifications to siRNAs. The most common types of nucleotide modifications among in-vivo therapies are multiple variants of the ‘morpholino,’ which alters the backbone of the siRNA to contain a six-membered ring in place of a pentose. This modification has many benefits, including increasing siRNA stability and membrane permeability. However, it is known that when the siRNAs are fully modified with these morpholinos, they are unable to interact with many aspects of the powerful endogenous RNA Induced Silencing Complex (RISC), including the endonuclease activity of Argonaute-2 (AGO-2), which restricts their mechanism of operation.

It has not yet been well studied how siRNAs behave when modified only partially with morpholino modifications, especially regarding their usage of the AGO-2 endonuclease function. Theoretically, a partial morpholino modification could confer some of the morpholino’s benefits of stability and increased membrane permeability, while still allowing the interaction with mRNA degradation pathway members, such as the RISC. In this study, it is demonstrated that siRNAs with thiomorpholino modifications at the head and tail of the siRNA can produce targeted protein knockdown in human cancer cells but are unlikely to utilize the AGO-2 endonuclease activity in-vitro, though further optimization studies are necessary. These findings will help inform the future design of siRNA with localized modifications for potential human therapies

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