Brown BIOL1220:Notebook/SynBio in Theory and Practice/Genes/Argonauts(miRNA pathway)

Background information
The argonaut family of proteins is one of the principal components of RISCs (RNA-induced silencing complex). The formation of these complexes is initiated by the Dicer enzyme that is also responsible for processing microRNAs (miRNA) – small, non-coding RNA molecules that do not manufacture proteins but downregulate gene expression by binding to complementary mRNA strands and performing endonuclease activity on them.

As well as being the site of miRNA-mRNA binding which then leads to translational repression, argonauts are partially responsible for the selection of the ‘guide’ strand and destruction of the ‘passenger’ strand. The guide strand, also known as the incorporated strand, is selected by the protein based on the stability of its 5’ end. The complementary or passenger strand is then degraded as a RISC complex substrate.

Several miRNAs have been linked to cancer and heart disease based on expression analysis studies that reveal disrupted miRNA expression in tumors compared to normal tissues.

They are also believed to play an important role in the control of diverse aspects of cardiac function and dysfunction.

Hence, the miRNA pathway that is facilitated by Argonauts is particularly important because of its role in downregulation. This process is essential, for example, when a cell is overly stimulated by a neurotransmitter, hormone or drug for a prolonged period of time and the expression of the appropriate receptor protein is decreased in order to protect the cell. Furthermore, the study of miRNA pathways are critical in encoding and understanding what actually happens to the genes that have been merely identified by genome sequencers.

From my research, these seem to be the specific argonaute proteins that have been identified by genome sequencers till date:
 * Argonaute 1 and its isoforms A, B and C
 * Argonaute 2 and its isoforms A, B and C
 * Argonaute 3 and its isoforms A, B, C, D, E and F
 * Argonaute 4
 * Argonaute 5
 * Argonaute 6
 * Argonaute 7

Sequence Alignments
For our research purposes, I hope to amplify DNA strands from Argonaute 1, which has been recognized as necessary for mature miRNA production that impacts on miRNA-directed RNA cleavage.

I have chosen aligned sequences of this protein as found in Drosophila melanogaster (House fly), Bombyx mori (silk worm) and Strongylocentrotus purpuratus (purple sea urchin).

[[Media:Argonaut 1 Sequence alignments.doc]]

Primer Details
Primer 1: AGO1A Amino acids: D M Q T S T M DNA sequence: GAY ATG CAR CAN WSN CAN ATG Primer sequence (5’ - 3’): GAY ATG CAR CAN WSN CAN ATG Melting temperature of primer: 47 - 56°C GC content: 43%

Primer 2: AGO1B Amino acids: E V W F G F H Q DNA sequence: GAR GTN TGG TTY GGN TTY CAY C Primer sequence is reverse complementary strand (5’ - 3’): GRT GRA ANC CRA ACC ANA CYT C Melting temperature of primer: 49 - 60°C GC content: 50%