Played by humans, scored by nature, online game helps unravel secrets of RNA

PITTSBURGH—Many video games boast life-like graphics and realistic game play, but have no connection with reality. A new online game developed by Carnegie Mellon University and Stanford University researchers, however, finally shatters the virtual wall.

The game, called EteRNA (http://eterna.cmu.edu) harnesses game play to uncover principles for designing molecules of RNA, which biologists believe may be the key regulator of everything that happens in living cells. But the game doesn't end with the highest computer score. Rather, players are scored and ranked based on how well their virtual designs can be rendered as real, physical molecules. Each week's top designs are synthesized in a biochemistry laboratory so researchers can see if the resulting molecules fold themselves into the three-dimensional shapes predicted by computer models.

"Putting a ball through a hoop or drawing a better poker hand is the way we're used to winning games, but in EteRNA you score when the molecule you've designed can assemble itself," said Adrien Treuille, an assistant professor of computer science at Carnegie Mellon, who leads the EteRNA project with Rhiju Das, an assistant professor of biochemistry at Stanford. "Nature provides the final score — and nature is one tough umpire."

Because EteRNA is crowdsourcing the scientific method — enlisting non-experts to uncover still-mysterious RNA design principles — it is essential that scoring be rigorous.

"Nature confounds even our best computer models," said Jeehyung Lee, a computer science Ph.D. student at Carnegie Mellon who led the game's development. "We knew that if we were to truly tap the wisdom of crowds, our game would have to expose players to every aspect of the scientific process: design, yes, but also experimentation, analysis of results and incorporation of those results into future designs."

The complex, three-dimensional shape of an RNA molecule is critical to its function. The goal of the EteRNA project is to design RNA knots, polyhedra and other shapes never seen before.

"We want to understand how RNA folds in a test tube and eventually in viruses and living cells," Das said. "We also want to create a toolkit of basic building blocks that could be used to construct sensors, therapeutic agents and tiny machines."

By synthesizing a design generated by game play, researchers will learn quickly whether the resulting molecule folds into the predicted shape, or something close to it, or if it even folds at all. Even designs that are not synthesized will be scored by nature, in that their scores will be based on the performance of similar designs previously synthesized.

"These experiments are the first-line strategy for validating a design and a crucial part of the scientific method," said Das, whose lab at Stanford synthesizes the molecules. "This makes EteRNA similar to what goes on in my lab on a daily basis: You make a prediction, do an experiment, make adjustments and start again." Initially, Das' lab is synthesizing eight designs each week, but is ramping up to synthesize about 100 a week.

RNA, or ribonucleic acid, long has been recognized as a messenger for genetic information, yet its role usually was overshadowed by DNA, which encodes genes, and by proteins, which do the work of the cell. But biologists now suspect RNA plays a much broader role as the regulator of cells, acting much like the operating system of a computer. Understanding RNA design could prove useful for treating or controlling such diseases as HIV, for creating RNA-based sensors and even for building computers out of RNA.

The game employs state-of-the-art simulation software that players use to generate designs. It includes training exercises and challenge puzzles for honing skills, as well as challenges for designing molecules that will be synthesized.

In its use of game play to generate results of scientific interest, EteRNA is similar to other online games such as Foldit, an online protein-folding game that Treuille helped create while at the University of Washington. In fact, Treuille and Das met when they sat at adjacent desks in the Washington biochemistry lab of David Baker, where Treuille was working on Foldit and Das was studying RNA and protein folding and occasionally offering advice.

Both men recognized that the lack of real-world feedback was a limitation of these games. They realized an RNA design game could solve this problem because RNA, unlike many biological molecules, can be readily synthesized in a matter of hours.

RNA consists of long, double strands of four bases — adenine, guanine, cytosine and uracil — with the shape determined by the sequence of the bases. The rules controlling shape are relatively simple, but the sheer size of the molecules greatly complicates the design process.

"We've already found it's better not to use regularly repeating sequences of bases because they prove unstable," Treuille said, based on play by beta testers. "We're trying to build things that work in nature, and nature favors solutions that are robust."

The game is integrated with Facebook, so players can post accomplishments to their Facebook wall automatically and can create groups that talk about play and compete with each other.

The first challenges are relatively simple, arbitrary shapes, Das said, but will soon begin to incorporate designs of scientific relevance, such as RNA switches that could be used to sense and respond to other molecules in living cells.

Ultimately, players may end up creating designs and making discoveries of their own. "They're already beginning to act like a scientific community," Treuille said. "One player solved a puzzle that a widely used algorithm could not. Another player has written a strategy guide that proposes an algorithm for solving design problems that is different and simpler than anything in the scientific literature."

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The EteRNA project is funded by a grant from the National Science Foundation.

For more information on EterRNA watch these video clips:
What is the EteRNA game? (1:19) http://wms.andrew.cmu.edu:81/nmvideo/eterna_1.mov
What have we learned from EteRNA? (1:57) http://wms.andrew.cmu.edu:81/nmvideo/eterna_2.mov
How was EteRNA created? (2:28) http://wms.andrew.cmu.edu:81/nmvideo/eterna_3.mov

About Carnegie Mellon: Carnegie Mellon University (www.cmu.edu) is a private, internationally ranked research university with programs in areas ranging from science, technology and business, to public policy, the humanities and the fine arts. More than 11,000 students in the university's seven schools and colleges benefit from a small student-to-faculty ratio and an education characterized by its focus on creating and implementing solutions for real problems, interdisciplinary collaboration and innovation. A global university, Carnegie Mellon's main campus in the United States is in Pittsburgh, Pa. It has campuses in California's Silicon Valley and Qatar, and programs in Asia, Australia, Europe and Mexico. The university is in the midst of a $1 billion fundraising campaign, titled "Inspire Innovation: The Campaign for Carnegie Mellon University," which aims to build its endowment, support faculty, students and innovative research, and enhance the physical campus with equipment and facility improvements.

Comparative study of the binding mode of 1IZI to HIV-1 protease with the binding mode of Indinavir to the wild-type HIV-1 protease.

Introduction

Human immunodeficiency virus, commonly known as HIV, is a retrovirus that causes AIDS. An enzyme called reverse transcriptase, found in this virus turns its RNA into DNA, and then the DNA is integrated into the host cell’s genome where viral RNA is transcribed and then translated into  a long polyprotein.

A protease is an enzyme which cleaves proteins to their component peptides. The HIV-1 protease hydrolyzes viral polyproteins into functional protein products necessary for viral assembly as well as subsequent activity. The HIV-1 protease is a homodimer containing 99 amino acids in each identical chain. An axis of symmetry is formed by each monomer in the active protease. Monomers are stabilized by aliphatic residues in hydrophobic core and dimer by noncovalent interactions, hydrophobic packing of side chains and interactions involving catalytic residues. There are two cysteine residues in each monomer but they do not make disulfide bonds (1). The active site is located between identical subunits with characteristic Asp25-Thr26-Gly27 sequence common among aspartic proteases. These two Asp 25 residues from both monomer act as catalytic residues. (2)

Aim and Objectives

The ligand for IZI will be identified from the X-ray structure and comparative analysis in binding mode of 1IZI and Indinavir to the HIV-1 protease will be made in this project. The amino acid residues in the active site and those involved in binding the inhibitor will be identified and their interaction behavior will be studied. The functional groups that correspond to the P1, P1', P2, P2', P1-P3 groups will help to know if the groups in 1IZI also bind in the same way. The alternative binding sites if present will be discussed in brief.

Methods

Protein Databank Europe (PDBe) (3), a project for collecting, managing and distributing macromolecular structure data derived from Protein Data Bank was used to get the experimental details of the protein structures using PDB Id. The pdb entry files (1IZI and 2BPX) were read in BODIL(4) and 1IZI was superimposed on top of 2BPX using VERTAA(5) within BODIL. The ligand of our structure (structure assigned for the project) was selected from Structure Editor tree and active site was viewed displaying only the area of 7.0 Å radius within the ligand molecule.

Result:

The inhibitor-HIV-protease complex structure (Figure 1) was a mutant type with mutations in three amino acid residues (A71V, V82T, I84V) which was found in the pdb entry file. The chemical formula for the inhibitor is actually C38H47N5O7.However, in the structure I got from PDBe (Appendix 2) it is C38N5O7 i.e with no hydrogen atoms. There are altogether 17 amino acid residues and a water molecule that are involved in binding of the inhibitor. Among them, 4 amino acids, ASP25B, ASP25A, ASP30B, GLY48B and a water molecule HOH1002A form hydrogen bonding. GLY27B and GLY27A form electrostatic interaction. Remaining 11 residues make van-der-waals interactions; those residues are LEU23B, LEU23A, ALA28B, ASP29B, ILE47B, GLY48A, GLY49B, GLY49A, PRO81B and THR 82A. Each of the atoms of the inhibitor that are involved in binding or can make interaction with the amino acids nearby are presented in the table (appendix 1). This result was derived from PDBe where the possible bond lengths below 4Å and interaction type was obtained.

The structure of our structure was superimposed on top of 2BPX structure (WT indinavir) and the ligands along with their neighbouring amino acids that were 7Å in radius were selected to study the interaction mechanisms.

 

Discussion

The PDB entry file has mentioned that there are only 3 mutations(A71V, V82T, I84V) in our structure(1IZI) however, one more mutation (SER37ASN) was observed while doing pairwise alignment. They might be missing in the PDB file because they are located in the peripheral loop of the structure. The water molecule that is conserved in the binding site (WAT2002) that is also responsible in binding the inhibitor to amino acid residue and is thus not replaced while binding to the inhibitor (appendix 2). The functional group for P2(alkyl) and P1’(phenyl) are same in both the inhibitors. P1-P3 group in indinavir doesnot have correspondence with any groups as it is extended a bit and on the other hand, 1Q50 is extended a bit in the opposite side. However, they have maintained similar conformation in between.

References

1. Miyeko M, David M (2001) HIV-1 Protease CLU Biology Department. (http://www.callutheran.edu/Academic_Programs/Departments/BioDev/omm/hiv_protease/molmast.htm)

2. Wikipedia (http://en.wikipedia.org/wiki/HIV-1_protease)

3.Velankar S, Best C, Beuth B, Boutselakis CH, Cobely N, Sousa Da Silva AW, Dimitropoulos D, Golovin A, Hirishberg M, John M, Krissinel EB, Newman R, Oldfield T, Pajon A, Penkett CJ, Pineda-Castillo J, Sahni G, Sen S, Slowley R, Saurez-Uruena A, Swaminathan J, van Ginkel G, Vranken WF, Henrick K, Kleywegt GJ (2010) PDBe: Protein Data Bank in Europe. Nucleic Acids Research, 38, 308-317

4. Lehtonen JV, Still DJ, Rantanen VV, Ekholm J, Björklund D, Iftikhar Z, Huhtala M, Repo S, Jussila A, Jaakkola J, Pentikäinen O, Nyrönen T, Salminen T, Gyllenberg M and Johnson M (2004) BODIL: a molecular modeling environment for structure-function analysis and drug design. J Comput Aided Mol Des 18(6),401-419

5.Johnson MS, Overington JP (1993) A structural basis for the comparison of sequences: An evaluation of scoring methodologies. J. Mol. Biol. 233: 716-738

Researchers to drill for hobbit history

Homing in on tooth cementum may help researchers extract DNA from Homo floresiensisremains.

Scientists are planning an attempt to extract DNA from the 'hobbit' Homo floresiensis, the 1-metre-tall extinct distant relative of modern humans that was unearthed in Indonesia, following a study that suggests problems in standard sampling methods in ancient-DNA research could have thwarted previous efforts.

This year, geneticists at the Australian Centre for Ancient DNA (ACAD) at the University of Adelaide hope to recover DNA from a roughly 18,000-year-old H. floresiensis tooth, which was excavated in 2009 from the Liang Bua site on the Indonesian island of Flores.

The premolar has been kept cold, and has been handled as little as possible to prevent contamination with modern DNA. But little, if any, of the ancient DNA is likely to have survived the heat and moisture of the tropics, and any that has may be highly fragmented.

Tony Djubiantono, director of the Indonesian National Centre for Archaeology in Jakarta, where the tooth is held, says that developments in DNA extraction techniques could overcome previous sampling problems, and have exciting potential for understanding the evolutionary history of H. floresiensis.

If the DNA can be extracted, comparing its sequence to that of other species could settle disputes over classification. For instance, Peter Brown, a palaeoanthropologist at the University of New England in Armidale, Australia, who described and named the species in 2004, is rethinking his initial classification. At first he put the species in the human genus Homo, but he now suspects that the hobbit's ancestors left Africa before Homo evolved so the species could belong to a different or new genus.

Source: Nature News