Muller, Ulrich
Evolution of catalytic RNAs, and the Origin of Life

Contact Information
Associate Professor

Office: Urey Hall 5218
Phone: 858-534-6823
Email: ufmuller@ucsd.edu
Web: www.ucsd.edu
Group: View group members
Education
2000 Ph.D., University of Technology Darmstadt, Germany
1995 BS, LMU Munich, Germany
Appointments
2014-present Associate Professor, UC San Diego
2006-2014 Assistant Professor, UC San Diego
2001-2006 Postdoctoral Researcher, Whitehead Institute, Cambridge, MA
Awards and Academic Honors
2021-2026
NASA research award
2020-2022
Cystic Fibrosis Foundation
2019-2022
NASA research award
2018-2020
Gilbert Gene Therapy Initiative
2016-2019
NASA research award
2013-2016
NASA research award
2012-2013
Hellman Fellow
2011-2012
Hellman Fellow
2008-2011
NSF research award
2004-2005
NRSA fellowship from the NIH
2001-2004
Postdoctoral award from the German Research Council (DFG)
Research Interests
The Muller lab is interested in catalytic RNA molecules (ribozymes), with two specific questions:

1 - During the emergence of life, how could catalytic RNAs have mediated self-replication and evolution?

The earliest evolutionary stages of life included a stage without encoded protein synthesis, and likely with RNA serving both as genome and as the only genome-encoded catalyst. Support for this idea comes from the findings that the ribosome (the protein translation machinery) is a catalytic RNA, that most cofactors are derived from nucleotides, and that lab-generated RNAs are able to catalyze many different chemical reactions. We are using in vitro selections to generate specific catalytic RNAs that could have been important in an RNA world. By characterizing their frequency in sequence space, their activity, and their dependence on cofactors, we are trying to find out how an RNA organism could have emerged from a prebiotic environment, and under which conditions this would have been most likely. Our long-term goal is to generate a self-replicating and evolving system based on catalytic RNAs in the lab. Such a system would by definition evolve into a more efficient replicator, and therefore guide our understanding of early, RNA-based stages of life. Additionally, evolving such a system in different chemical environments (for example, in the presence of amino acids) may show how such an early stage could have evolved into more sophisticated systems.

The focus of our current work is on ribozymes that use the prebiotically plausible molecule cyclic trimetaphosphate (cTmp) as energy source. This molecule could have supplied the energy that is essential for the self-replication of an RNA organism. In a proof-of-principle study, we used an in vitro selection from more than 10^14 different, random sequences to identify ribozymes that can catalyze the triphosphorylation of RNA 5'-hydroxyl groups using trimetaphosphate. These results showed that ribozymes would have been able to use trimetaphosphate as energy source for early life forms.

To generate an early metabolism, ribozymes are central that generate chemically activated nucleotides - such as NTPs. A direct in vitro selection of such ribozymes is technially extremely challenging because it requires the ribozyme to bind two small molecule substrates and catalyze their reaction, and because the product of the reaction is a freely diffusing molecule. To address this challenge we developed a system that selects a new ribozyme from random sequence in emulsion. In 10^16 droplets of a water/oil emulsion, we dispersed 5 x 10^15 library molecules, and used a second ribozyme to tag library molecules that generated the NTP. After several rounds of selection, the selected RNA sequences were analyzed by High Throughput Sequencing analysis, and catalytically active sequences were ideantified in biochemical assays. The result was a ribozyme that generates GTP from guanosine and cTmp with a catalytic rate enhancement of 18,000-fold. The turnover is very low (1.7-fold), and we are currently working to increase it such that a self-replicating system can be supported.

We are also pursuing additional projects with the long-term goal of generating a self-replicating and evolving system of RNA molecules in the lab. Because such a system would consist of only a handful of catalysts it would allow us to completely understand a life-like system based on all mutual molecular interactions, and it would allow us to follow their evolution over time, and better understand the origin of our distant ancestors.

2 - Can catalytic RNAs be used to treat genetic diseases by repairing the mutations on the RNA level?

Natural group I intron ribozymes are cis-splicing, which means that they remove themselves from the primary transcript in two transesterification reactions. These cis-splicing ribozymes can be transformed into trans-splicing ribozymes. In that new format, the ribozyme can be used to repair genetic mutations on the RNA level. To be therapeutically useful the efficiency of these ribozymes needs to be increased. We are doing this by identifying the best splice sites on target RNAs, and by evolving the ribozymes for high activity in cells.

In related work we have re-engineered the ribozyme to splice on two splice sites. These spliceozymes recognize a target RNA at two splice sites, remove the intervening sequence, and join the two flanking sequences. Because this is analogous to the spliceosome we have termed these ribozymes 'spliceozymes'. We have evolved these ribozymes in bacterial cells for higher efficiency. The resulting ribozymes generate much more of the product sequence by a subtle re-balancing of the activities at the 5'-splice site and 3'-splice site. This re-balancing leads to a much lower formation of side products and consequently a more efficient conversion to the desired product.
Primary Research Area
Biochemistry
Interdisciplinary interests
Macromolecular Structure
Cellular Biochemistry
Bioorganic

Outreach Activities
CAMPUS EFFORTS

Advisory Service - Participant in developing the GE curriculum at Thurgood Marshall College in 2009. Thurgood Marshall College places an especially high importance on promoting diversity, for example in its specifically designed program Dimensions of Culture (DOC).

Recruitment Efforts - Assist in the recruitment efforts of the Thurgood-Marshall College, in two recruitment seasons.

Mentoring Efforts - Involvement in the Thurgood-Marshall mentorship program for transfer students, specifically aimed at helping disadvantaged transfer students.

COMMUNITY EFFORTS

My lab is dedicated to supporting an equal opportunity environment. This is reflected in the numbers of students in my lab: Three of the seven PhD students from my lab who have so far defended their thesis are female. Five of twelve undergraduate researchers who worked in my lab were female, and five of them were from an ethnic background (Asian/Hawaiian/African American).

From 2018 to 2020 I served as Vice Chair for Education, and since 2020 I am serving as Vice Chair for Graduate Education in the Department of Chemistry & Biochemistry. Both roles served the needs of students on many different levels. In my current role as VCGE I am addressing the needs of departmental graduate students in many different forms, including information sessions, meetings to solve specific problems, and a regular 'tea hour with VC Uli' to address any challenges faced by graduate students.
Image Gallery


In emulsio selection for a GTP synthase ribozyme. (A) Schematic of the selection system (B) DLS analysis of the emulsion (C) Kinetic Analysis of GTP synthesis..

Coupling of GTP synthesis with RNA polymerization in a minimal metabolic system,


Secondary structure of the GTP synthase ribozyme, as determined by chemical probing.

Selected Publications