Biomod/2014/VCCRI/Project/Problem

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EchiDNA 2014

Background

If you ever start trying to design a DNA nanomachine so small you could fit a few thousand billion of them inside a grain of sand, it might be a cool idea to ask someone who’s tried it before!

Turns out Mother Nature has been testing out all sorts of designs for the past few billion years. Our team has taken inspiration from the Bacterial Flagellar Motor (the BFM). The BFM is a dynamic protein complex that self-assembles in the cell membrane of bacteria. It exploits a gradient of hydrogen or sodium ions to generate rotation at over 100,000 rpm. The ring of proteins responsible for generating torque also acts as a sensor and binary switch, changing the direction of rotation the flagellar motor depending on the environment. This sensing mechanism is also tuneable, allowing bacteria to act appropriately in different environments by adapting the concentration threshold that triggers rotational switching the BFM.

 

Our team has built a DNA nanomachine inspired after the tuneable, binary switching mechanism of the BFM. We believe this project lays the groundwork for a new kind of DNA biosensor and also provides a novel experimental system in which to understand the machinery of life.


Cooperativity

How does humankind build cities, empires, or the wonders of the world? We cooperate. How does Nature build a human? Cooperativity.

In its essence, cooperativity is the phenomenon where communication between individual elements leads to strikingly different group-based behaviour, such as collective decision making. As esoteric as it sounds, it is a relatively intuitive concept to illustrate:


Consider a colony of ants. How do they decide to move? If each ant acted completely independently of the others, you would have difficulty getting the whole colony to move in a coordinated manner, with ants scrambling everywhere. This is non-cooperative behaviour. On the other hand, if the ants talk to each other, and they make the decision together through gossip and pheromone trails, the whole colony can decide as a group whether or not to move. This is cooperative behaviour. This is how flocks of fish and birds decide which direction to move, how entire species decide when or to migrate, and how bees build organised hives.
The four subunits of hemoglobin bind oxygen molecules cooperatively.


Cooperativity exists at all scales of nature. A classic example of molecular cooperativity is the hemoglobin in our blood. This vitally important molecule has the capacity to bind four molecules of oxygen to four binding sites. For hemoglobin, cooperativity means that the last oxygen molecule binds much more readily than the first oxygen molecule due to conformational changes of the protein induced by the first oxygen molecule binding. However, it is being realised that more and more cellular processes are driven by cooperativity.

Switching mechanism of the BFM.
For instance, how does the BFM exhibit binary switching between clock-wise and anti-clockwise rotation? How is it possible for a protein in one part of the nanomachine to communicate with a protein on the other side? How can molecules have coordinated behaviour over such distances? One model suggests that the protein sub-units exhibit cooperativity.

Our team has designed a unique, DNA-based system which mimics the switching mechanism of the BFM. We are building a unique experimental system from scratch in which we can explore the phenomenon of cooperativity.
 

Molecular Beacons

The first step towards building a cooperative biosensor is designing the single switch. This is the repeated sub-unit that exhibits a detectable conformational change and can be tethered to its neighbours. And it turns out that our simplest designs of this sub-unit actually resemble an existing DNA nanotechnology - the molecular beacon.

How do we tell if a particular DNA sequence is present? This posed a great problem that took some brilliant minds to crack. Tyagi and Kramer offered the solution of molecular beacons: synthetic constructions that were able to report the presence of a DNA strand with a particular sequence. Their design still forms the basis for many of the newly developed molecular beacons today.

Molecular beacons have four main components:
  • A loop region that is complementary to the target sequence,
  • Two regions that are complementary with each other, closing the loop into a hair-pin,
  • A fluorophore at one end of the sequence,
  • And a quencher at the other end of the sequence.

Its operation depends on two major principles: DNA hybridisation and Förster resonance energy transfer (FRET). An understanding of DNA hybridisation is important because the hairpin DNA must be designed to preferentially bind to the target DNA by unwinding the native hairpin structure. FRET is important as it provides the means of interrogating the state of the hairpin DNA.


In its native state, the molecular beacon contains the fluorophore and quencher in close proximity due to the hairpin structure. According to FRET, this means no fluorescence will be seen from the fluorophore, as it will all be absorbed by the quencher.

In its bound state, the molecular beacon contains the fluorophore and quencher far away from each other. According to FRET, this means fluorescence will be detected.

The molecular beacon is an awesome innovation that non-destructively interrogates for the presence of a target DNA sequence. However, molecular beacons are limited in two key ways -
  • As the length of the target sequence increases, the probability that mismatched DNA opens the switch also increases.
  • Most applications rely upon PCR amplification of the target DNA. This increases the likelihood of false positives through mutations during PCR and also makes quantification of the initial environmental sample difficult.
Our team is exploring a new kind of dynamic biosensor by building on the proven technology of molecular beacons and the sophisticated switching mechanism of the BFM.

Relevance

Our project is relevant for two fundamental reasons:
  • We anticipate that our design will prove to be a novel biosensing technology by exhibiting a tuneable binary output.
  • This project could lead directly to scientific discoveries because we are building an entirely new experimental system in which we can answer basic questions about the thermodynamic and kinetic consequences of cooperativity.
Our team is building a new kind of biosensor inspired by nature. We have essentially tethered together a ring of molecular beacons in an analogous fashion to the protein sub-units of the switching mechanism in the BFM. This project not only allow us to fully characterise a novel cooperative molecular biosensor, but also provides insight into the design principles of dynamic bionanotechnology.


Click here to continue on to our 'Approach' page


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