How does "Programming cell-free biosensors with DNA strand displacement circuits" work?

So how does this genetic logic gated system work?

First, we need to cover some molecular biology basics.

DNA – transcription – translation

DNA – usually found as a double stranded molecule and depicted as a series of letters – As Gs Ts and Cs.?One strand has a particular sequence of these letters (not letters in real life!) and the other strand has a corresponding sequence that binds to (hybridises to) the first strand. As bind to Ts; and Gs bind to Cs. A G binds to a C more strongly than an A binds to a T.

DNA is generally thought of as the thing that encodes our proteins – proteins are the molecules in our cells that do all sorts of things.?The order of the As Gs Ts and Cs determine which protein is made.

To make a protein from DNA, the order of the As Gs Ts and Cs in the DNA is converted into another molecule – RNA.?RNA is similar to DNA, and to make a protein the sequence of the RNA is converted into a particular sequence of amino acids – the building blocks that make up proteins.

So the order of the GATCs in the DNA determines the order of the GATCs in the RNA (though RNA uses a U instead of a T!), and the order of the GATCs in the RNA determines the order of the amino acids in the protein, and the order of the amino acids in the protein very much determines the structure and function of the protein. So our DNA encodes all of our proteins – and tiny changes in our DNA give changes in the proteins, and are what makes me me, and you you.

The conversion of the DNA sequence into RNA is called transcription; and the conversion of the RNA into protein is called translation.

A chunk of DNA that encodes and RNA that encodes a protein is what we call a gene.

But – not all RNAs are used to make proteins – so called non-coding RNAs. ?RNAs have been found to have all sorts of functions themselves, beyond encoding proteins.?They can be used to silence or turn off genes ?- so that the protein product isn’t made.?They can have catalytic activity, like protein enzymes. And they can have very specific binding affinities, like antibodies.??Non-coding RNAs are one key part of the system in this paper.

Transcription factors

Transcription (the reading of the GATCs in the DNA so that they are converted to RNA) from a piece of DNA is usually started from a promoter – a promoter being just another piece of DNA that certain proteins can bind to.?Some of these proteins are called transcription factors, and they can turn on (an activator) or turn off (a repressor) transcription.?

Some transcription factors can only turn on or off transcription when they bind to a particular molecule – or ligand. The ability of transcription factors to be active or inactive in the presence or absence of a particular ligand is the basis of the sensor.

Toe-hold mediated strand displacement

A lot of words – but it basically describes the ability of one nucleic acid molecule (such as a strand of DNA or RNA) to push off, or displace, another strand.

As we said, DNA typically exists as a double stranded molecule.?In many cases each of the two strands in the double stranded DNA molecule are the same length, giving blunt ends.?But, it is possible to create double stranded DNA where one strand is shorter than the other.?This leaves an overhang – where the first strand is a bit longer than the second strand and has no other strand to bind to.?This is called the toe-hold.

If we add a third single strand of nucleic acid– it could be a single stranded DNA or a single stranded RNA, and if part of that strand has a sequence (the GATCs) that matches the sequence of the toe-hold overhang (of the first strand), the third strand can bind to the over hang and push off the second, shorter strand of DNA from the original double stranded DNA molecule.

One of the clever bits is that this second strand, the one that is pushed off can go off and do other things.

Reporter

The other key part of the system is a reporter.?This is something that tells us the answer and that we can detect – is the test ligand there, or not? What is the concentration of the test ligand?

The reporter in this case is another bit of double stranded DNA (remember, the strands are bound to each other via the G/C and A/T pairing) – one strand has a fluorescent molecule (strand F) an which gives off light that we can detect, but the other strand has a quencher molecule (strand Q), which stops us being able to see the light from the fluorophore.?When the two strands are bound together in the double stranded DNA molecule, we can’t see any fluorescence – it is quenched by the quencher.?The only way we can detect fluorescence is if these two strands are separated – once the duplex is broken, the quencher can’t quench the fluorescence, and we can see the light.

So, somehow the system needs to be engineered so that the strands are separated when the target ligand is present.

Putting it all together..

In the simplest example:

·??????If the test ligand is present, it binds to a particular transcription factor (that is included in the cell free system)

·??????Once the transcription factor binds to the test ligand, it can bind to a promoter and initiate transcription from a chunk of DNA, and produce a non-coding single stranded RNA molecule.?In the paper, this RNA molecule is called InvadeR

·??????In the cell free system, we also have another double-stranded DNA molecule with a toe-hold that matches part of the sequence of InvadeR; and also has a sequence that matches the reporter DNA molecule.

·??????The InvadeR RNA binds to the toe-hold and pushes off the shorter of the two DNA strands.

·??????The shorter of the two DNA strands binds to the reporter and pushes off the quencher labelled strand (or pushes off the flurophore labelled strand – same effect) – and we get light that we can detect!

The advances of this paper are largely in the use of toe-hold mediated displacement.

A range of different molecules can be combined – for example the second strand that is displaced by InvadeR could itself be a second InvadeR molecule that invades a second toe-hold fragment.

The sequence and length of the toe-holds can be modified. ?Remember, the GC pair is stronger than the AT pair – this means that if we alter the ratio of GC to AT in the sequence, we can affect the binding affinity of InvadeR and the toe-hold, which can affect the speed of the reaction.?

The authors of the paper also altered the length of the toe-hold – InvadeR RNA binding to a longer toe-hold is more energetically favourable than binding to a shorter toe-hold.?This lead to the development of a “threshold gate”.

The threshold gate is another DNA molecule with a longer toe-hold than a toe-hold fragment that contains the DNA molecule that will ultimate bind to the reporter (called the signal gate). Since binding of the InvadeR RNA to the threshold gate DNA molecule with the longer toe-holds is more energetically favourable than binding to the shorter toe-hold of the signal gate, the invasion of InvadeR RNA into the signal gate, with the shorter toe-hold, only occurs once all of the threshold gate DNA molecules have been invaded.?The threshold-gate effectively acts like a sponge – soaking up and slowing down the ability of InvadeR RNA to bind to and invade the signal gate DNA molecule.

By using different concentrations of threshold gate, it is possible to get a semi-quantative readout of the concentration of the test ligand (low concentration of ligand will produce a low amount of InvadeR RNA and will need a low concentration of threshold to have enough InvadeR RNA left to invade the signal gate; a high concentration of ligand will make lots of InvadeR RNA and so would show a signal with a low or a high concentration of threshold.