Friday, September 14, 2012

More Potentially Revolutionary Developments in Gene Expression

In "Steps Toward the Mastery of DNA Based Life" we looked at amazing developments in manipulating DNA transcription. Today we will look at an intriguing development in designing novel functioning RNA. It is important to remember that while DNA holds the instructions for creating and maintaining a functioning organism, it is RNAs -- along with specific proteins -- that do most of the hard work. That means that the ability to design and manipulate RNA function may provide us with the quickest route to a powerful control of gene expression.

More on the new development:
For synthetic biologists a key goal is to use RNA to automatically engineer synthetic sequences that encode functional RNA sequences in living cells. While earlier RNA design attempts have mostly been developed in vitro or needed fragments of natural sequences to be viable, scientists at Institut de biologie systémique et synthétique in France have recently developed a fully automated design methodology and experimental validation of synthetic RNA interaction circuits working in a cellular environment. Their results demonstrate that engineering interacting RNAs with allosteric behavior in living cells can be accomplished using a first-principles computation.

...Drs. Alfonso Jaramillo, Guillermo Rodrigo, and Thomas E. Landrain had to address several challenges in their study. "It is common practice – and unavoidable – to use computational algorithms to aid in the design of RNA molecules," Jaramillo tells For example, he illustrates, computing minimum energy conformation, since one single nucleotide can stabilize an alternative conformation. Until now researchers have used computer assisted design to design synthetic RNAs that could combine functional fragments from known RNAs.

...This evolutionary computation technique relies on mimicking the relevant steps of natural evolution, that is, the iterative improvement of a given solution by using selection. "However, we don't have to be slaves of analogy and are free to consider what we think is more relevant to our problem," Jaramillo points out. "We would start from a random sequence and would randomly modify it by applying simulated annealing techniques, implemented by a Metropolis Monte Carlo algorithm," which solves a problem by generating suitable random numbers and observing that fraction of the numbers obeying some property or properties. "Contrary to natural evolution, our walks would not be completely adaptive but we could allow a decrease in fitness. We aim at the engineering of an ensemble of RNA species that could interact in a predefined way. Our first challenge was that in living cells, such molecules are very prone to degradation if they do not have a stable structure."

...After publishing the PNAS manuscript, Jaramillo adds, the team further validated the orthogonality (the ability to selectively translate mRNA) of their RNAs in E. coli. They're also constructing a XOR gate device working inside the cell – something never done in bacteria and just recently achieved in mammals1.

The researchers are planning to extend the methodology to include the RNA-small molecule interactions and the incorporation of known functional RNA sequence fragments (such as ribozyme sequences) to create complex RNA interactions never seen before. "We've already succeeded in experimentally validating in E. coli a new type of such an interaction, consisting of an inactivated riboregulator that could be activated by a ribozyme after the introduction of a small-molecule inducer."In this type of reaction, the number of different species is not conserved, as after the introduction of the inducer we get a RNA cleavage. "We've named this new riboregulator-ribozyme chimera a regazyme, and have also validated the full design of a riboswitch.

Jaramillo also notes that other research might benefit from their findings, including the high-throughput design of new regulators for large-scale engineering projects. "Also, we can foresee using allosteric RNAs to sense mRNAs by being subject to a conformational change after binding that could trigger a reporter." This would open the way to genetically-encoded and non invasive monitoring of gene expression dynamics – an important and unmet challenge in biophysics. "We're also exploring the use of RNA," Jaramillo concludes, "to create artificial signal transduction cascades."
These are interesting and powerful ideas. Not only can this technique be used to provide new levels of control and monitoring of gene expression -- they can also be used to create new types of computational devices using biological materials.

Study abstract in PNAS

Using DNA, RNA, or proteins to create logic circuits in cells may seem like a waste of time when we already have such powerful silicon-based computational devices. But the ability to embed designed computation at the cellular level provides humans with a level of explicit and specific fine control over biological functions which was only dreamed of in the past.

And it is likely that we will need that level of control to accomplish the conquest of cancer, degenerative disease, and ageing -- to say nothing of our quest to grow ever wiser and brighter.


Friday, September 07, 2012

Encode Project Opens Unimagined Vistas of Discovery

The more we learn about the mechanisms of life, the more we discover there is yet to be learned. Results from the "Encode Project" have reminded us of how little we know about the human genome and epigenome -- while at the same time opening the door to previously unimagined possibilities for discovery and scientific advancement. With this new opportunity for acquiring vast new knowledge, the road to longer lifespans and better brains has just gotten wider, smoother, and more solid.

We are just beginning to learn how our genes are regulated on a moment to moment basis. If we are to achieve our goals for longer, more capable, and more fulfilling lives, we will have to use all of our wits and tools -- and develop a lot of new ones.
When the Human Genome Project revealed that only around two percent of the genome is made up of protein-coding genes, it was suggested that the rest was made up of "junk DNA". The unsatisfactory conclusion left many geneticists skeptical, and Encode's findings now prove beyond doubt that the theory was way off mark.

The new research instead shows that 80 percent of the 98% unaccounted for has some kind of biochemical function, with 10,000 genes tasked with regulating the DNA responsible for coding proteins -- these 10,000 are responsible for building single-strand RNA molecules that regulate the 20,000 protein-coding genes. The mass of otherwise unaccounted for DNA actually represents a series of around four million "switches" that regulate other genes, and around nine percent of DNA helps code these switches (the figure could end up being nearer 20 percent, however).

...Encode has its work cut out. Having identified these millions of genes and investigated what turns them on an off, the team needs to track how they are connected and which genes they control. This will prove particularly challenging, considering genes in the three-dimensional genome are not mapped out in a straightforward manner -- a gene-controlling switch could be located somewhere entirely different from the gene it controls. Understanding the complex circuit route is key to understanding the human genome while, according to Birney, identification of the entire genome is only about 10 percent done.


A User's Guide to the ENCODE Project (PDF)

First came the Human Genome Project, focusing on protein-coding genes. Next came the Encode Project, focusing on genes that regulate other genes. Now we will have to learn how the entire complex works together, for the sake of a better human future.


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