The Bartel and Szostak experiment aimed to show that chemical selection could occur in an RNA world. They synthesized a pool of random RNA molecules and tested their ability to catalyze the ligation of short RNA tags. Molecules that showed catalytic activity were selected and amplified to produce successive pools. Testing showed that the catalytic activity increased over 10 generations, supporting the hypothesis that chemical selection could drive the evolution of RNA enzymes and supporting the idea of an early RNA world.

1. Maria Chiaffarano Bartel and Szostak Experiment

2. HOW DID LIFE BEGIN?

3. Four Overlapping Stages Scientists used four stages to understand the origin of life Stage 1 Nucleotides and amino acids were produced prior to the existence of cells Stage 2 Nucleotides became polymerized to form RNA and/or DNA and amino acids become polymerized to form proteins Stage 3 Polymers became enclosed in membranes Stage 4 Polymers enclosed in membranes acquired cellular properties

4. Chemical Selection What is chemical selection? Chemical within a mixture has special advantageous properties Properties allow it to increase in amount Hypothesis The cellular characteristics that exist today evolved from an “RNA world”

5. RNA World Scientists believe that the world used to consist of RNA based organisms Studied the building blocks Amino acids and nucleotides

6. Key Functions of RNA RNA has three key functions that encourage scientists to favor it as the first macromolecule found in protobionts Ability to store information in its nucleotides Nucleotide sequence has the capacity for self-replication RNA has many enzymatic functions Act as ribozymes

7. ADDITIONAL ADVANTAGES OF RNA DNA and proteins are not as versatile as RNA DNA has limited catalytic activity Proteins do not undergo self-replication However: RNA can perform functions that are characteristics of proteins while simultaneously serving as genetic material with replicative and informational functions

8. Bartel and Szostak Experiment David Bartel and Jack Szostak First study that used RNA molecules with a particular function (1993) Synthesized a mixture of 10^15 RNA molecules also known as “long RNA” First region (5’ end) constant region among all “long RNAs” (identical) Second region, variable region (220 nucleotides) Hypothesized that the variable region could possibly result in long RNA with the enzymatic ability to catalyze a covalent bond between two adjacent nucleotides

9. Materials Many copies of short RNA Had a tag sequence that binds tightly to “beads” (column packing material) Had a complementary sequence to a site in the constant region of the long RNA No variable region, all the same Long RNAs with the constant and variable regions Variable regions were made using a PCR step (caused mutations in the region)

10. Overview of the experiment

11. Step 1 The long RNAs and short RNAs were incubated together Promote hydrogen bonding Time was given for the molecules to form covalent connections (only if the RNA had the enzymatic ability to form covalent bonds) The long RNA molecule variable regions may rarely have enzymatic ability to connect the 3’end of short RNA to 5’end of long RNA

13. Step 2 The mixture passed through a column of beads The beads would bind to the tag sequence of the short RNA only! Tag sequence promoted the binding of the short RNA to the beads IF the long RNA had the ability to bind to the short RNA, it would also be attached to the beads Additional liquid was added to filter out the long RNAs that did not covalently bond to the short RNAs

14. Step 3 The main purpose of this step was to get rid of the beads Low pH solution was added in order to prevent the tag sequence from binding to the beads The tightly bound RNAs are flushed out of the column They were flushed into a flask and labeled “Pool #1” Beads were left behind

15. Step 4 Pool #1 was used to make a second batch of long RNA molecules Polymerase chain reaction (PCR) was used Variable regions were derived from the variable regions of pool #1 RNA molecules, expected to have enzymatic activity Reverse transcriptase was used to make cDNA PCR primers recognized beginning and end of the long RNA sequence and copied only this region cDNA used as a template to make long RNA via RNA polymerase

16. Steps 5 & 6 The procedure was repeated in order to generate 10 consecutive pools of RNA molecules A sample of the original population (Pool #1) and each of the following 10 pools were collected in order to test for the enzymatic ability to catalyze a covalent bond between adjacent nucleotides

18. Conclusion Data: Chemical selection is possible Scientists knew this because the experiment showed that there was an increase in covalent bond formation from pool 1 to pool 10 In each generation, the catalytic enzyme activity increased Pool #10’s enzymatic activity was approximately 3 million times higher than the original random pool of molecules