Section 12: SELEX and RNA evolution in vitro; Riboswitches and Aptamers

What has SELEX successfully been used to produce?

1. RNA aptamers that act like antibodies from a single amino acid to long polypeptides 2. RNA with novel functions 3. RNA with novel enzymatic properties

What are the 2 techniques combined in SELEX?

1. The ability to mutagenize DNA WITH 2. The ability to select or enrich for a particular RNA activities

What are the 2 domains of riboswitches and their functions?

1. sensor domain: acts as a aptamer; binds a small molecule 2. effector domain: causes a change in gene expression pattern

What is SELEX used for?

A method for directed evolution of novel RNA structures and enzymatic activities

This Aptamer is then attached to the ____ Domain and a second round of SELEX is used to make the riboswitch. The Riboswitch is then attached to a _____, for example GFP. When the ligand concentration changes then the Riboswitch rapidly transduces the information into a change in GFP _____.

Effector reporter intensity

SELEX TECHNIQUE(1): Essentially, a pool of single stranded DNA oligos is produced by chemical synthesis in which ____ bases are a single unique sequence (they are held constant) while a middle region consists of ____ sequences. The "complexity" of this pool of DNA is determined by the ___ of the randomized region which in this case is 30 nts long. Therefore this DNA pool contains 430 = 1018 unique DNA sequences and is also referred to as a "____ ____".

Flanking random length sequence space

Synthetic riboswitches are being and have been developed where the Aptamer Domain was developed by using ____.

SELEX

What is SELEX an acronym for?

Systematic Evolution of Ligands by Exponential Enrichment

What is a riboswitch?

a segment of RNA that senses changes in the environment that leads to changes in gene expression

SELEX TECHNIQUE(4): A mutagenic variation step can be introduced by using nucleotide _____ during steps 5 and/or 6 or a variant ____ ____enzyme (or variant reverse transcriptase) that has a high error rate.

analogues Taq polymerase

SELEX TECHNIQUE(2): Using PCR and in vitro transcription, an ____ RNA copy is made from this DNA pool. The RNA pool then undergoes a selection process in which those RNA sequences able to perform a given function, ie the "_____" are pulled out of this mixture. The remaining RNA sequences are discarded, ie. the "____".

identical Winners Losers

Riboswitches transduce ligand binding into genetic regulation due to a _____ of the shape of the RNA that is a Riboswitch is a "shape sensing" RNA.

remodeling

SELEX TECHNIQUE(3): The RNA Winners are then _____ _____back into a DNA sequence pool, which is now enriched for RNAs with the desired properties. Multiple rounds of this selection process results in only a few ____ sequences which then can be analyzed individually

reverse transcribed Winner

What is an RNA aptamer?

short RNA that binds tightly to a target molecule and can be either natural or artificial as identified by SELEX methodology

What is an aptamer?

short nucleic acid sequences or short polypeptide molecules that bind to a specific target molecule analogous to an antibody binding to a lingand

An example of riboswitching is in the thiamine biosynthesis operon where build up of the final product, thiamine, results in thiamine binding to a riboswitch in the mRNA of the first gene in the metabolic pathway and this binding _____ _____ translation of that mRNA and hence the first gene is ____ ___ and so the pathway is______ ___.

shuts down shut down turned off