
\blankpage

\chapter{Experiment: Storing Clocked Programs in DNA}
\label{ch05}

\section{Constructing stored instruction stacks}

\subsection{Introduction}

In order to demonstrate that instructions 
can be stored within DNA\footnote{Aidan Daly carried out
the work in this chapter in the summer of 2010 and wrote most of this 
description.}
\index{Daly, Aidan}%
later to 
be released with clock strands, we created instruction stacks of various 
lengths.
Constructing stacks is very similar to 
deconstructing them in that it involves pouring in alternating strands.
\index{stack construction}%
For construction, however, we would start with the ``base'' strand, and 
alternately pour anti-tick and instruction strands.
This method, though 
time-consuming, proved to be the most reliable for making stacks of a 
determined length, and unlike many one-step methods, would allow for the 
construction of color-changing stacks. 

Construction time is not a major concern
because stacks can be prepared far in 
advance of their use and stored stably at low temperatures.
In addition, 
construction (program compilation) 
can easily be automated, which would make it even more 
convenient.

Our findings indicate that this method
of construction should be a viable 
method for making a collection of
stacks all of the same approximate length. We have found
that stoichiometric 
\index{stoichiometry}%
balancing of input strands can 
curb runahead chain reactions.
Such chain reactions could occur when
excess anti-tick and instruction strands continue to hybridize and create 
longer stacks than intended.
\index{runahead stacks}%
As we have discussed in Chapter~\ref{ch04},
stochiometric balancing (perhaps in coordination
with a flushing mechanism) is also useful for the 
deconstruction step, again to counter runahead stacks.
Each 
construction/deconstruction step requires
slow cooling steps for accurate 
hybridization of added strands, but, again, construction time is not a major 
issue.

\subsection{Overview of methods}

We first tried a one-step method,
which combined base strands, 
instructions strands, and anti-tick strands
in a 1:4:4 ratio to see if we 
could create stacks consisting of approximately four instructions.
In this 
approach, we combined all the strands
at 40\r{ }C to disrupt any unwanted 
base pairing. These pairings usually occur over small ranges (2-3 
\index{base pairing}%
nucleotides) between strands during storage at low temperatures, when it 
becomes favorable for any sort of interaction to form between strands.
We 
then cooled the combined strands slowly, as slow cooling favors correct 
hybridization between the longest matching sequences.

We ran the resulting construct on
a native gel (one that allows DNA to 
\index{gel}%
exist in a double stranded state, thus not disrupting our constructs as 
would be the case for a denaturing gel). 
\index{denature}%
A gel permits us to determine the length of single and double strands.
If there is just one size, there will be a single bar corresponding to that
size. When there are many sizes, there will be a smear, where each
part corresponds to one of the sizes.

The results (see Figure~\ref{ch5-fig01})
showed a range of sizes, indicating
that this one-pot method yielded 
many chain reaction constructions.

\begin{figure}[hbt]
\begin{center}
	\includegraphics{ch5-fig01.eps}
\end{center}
\caption{ 
In this one step construction method,
the stack appears as a large smear in both of the lanes we ran it in, 
indicating the presence of many differently-sized constructs, containing 
anywhere between one and four instructions.
The one step construction method is therefore insufficient if we
want all stacks to be of the same length.}
\label{ch5-fig01}
\end{figure}

For this experiment, we used a small (8
bp), single-stranded base strand. 
The anti-tick strand was a 16bp single
\index{tick}%
\index{anti-tick}%
strand of DNA, and the instruction 
was a 56bp hairpin.
\index{hairpin}%
Each of these components was run in an independent lane 
on the gel as a reference for size.
The base strand was too small to appear 
on the gel and would dissociate
from the stack fairly easily due 
to its small region of complementarity.
The stack appeared as a large smear 
indicating the presence of many differently-sized constructs, containing 
anywhere between one and four instructions.

The smearing suggested that a 
sequential construction technique would be better. 
Sequential construction entails
alternately pouring and hybridizing balanced 
amounts of anti-tick and instruction strands
in order to control 
the length of the stack.

Because a sequential approach requires
multiple rounds of heating and 
slow cooling (one for each strand to hybridize),
we had to design a larger base 
strand.
The single-stranded 8 base pair fragment would not complement a 
large range, and would denature (i.e. the two complementary strands 
would split apart) from the
bottom of the stack at relatively 
low temperatures.
Thus, we designed a new base as a composite of two strands 
-- SB (small base) and LB (large base)
-- with an overhang that bound to the 
first instruction of the stack.
This increased size also allowed us to 
visualize the base on a gel.

Because we were performing a sequential
construction, we developed a naming 
system to categorize all intermediates
we would hope to observe on the gel (see Figure~\ref{ch5-fig02}).


\begin{figure}[hbt]
\begin{center}
	\includegraphics[width=\textwidth]{ch5-fig02.eps}
\end{center}
\caption{ The results of a sequential construction method: precise
selection of lengths.
}
\label{ch5-fig02}
\end{figure}

The sequential addition method consisted of the following protocol:
\index{sequential addition method}%
heat
equal molar amounts of 
the two base strands to a high temperature then cool 
slowly to allow hybridization.
For each subsequent step, heat the current 
intermediate to a temperature high enough to disrupt 
unwanted base pairings, but not high enough to denature the construct.
For 
example, when forming intermediate \textbf{C}, we combined 
an equal molar amount of 
anti-tick and \textbf{B}
in a vessel and heated to just 
below the temperature when the top instruction
strand of \textbf{B} would 
dissociate.
These temperatures were determined by the length of 
complementarity between the strands, which
in this case was 8 base pairs, as 
well as the GC content of the strands.
The resulting mixture would then be 
slowly cooled to allow desired hybridization.

Following this method, we constructed
\textbf{A} through \textbf{C }and ran 
the results on a native gel (see Figure~\ref{ch5-fig03}.).

\begin{figure}[hbt]
\begin{center}
	\includegraphics{ch5-fig03.eps}
\end{center}
\caption{Results of the first 
attempt at a sequential addition method on a native gel -- less of 
a smear but still not a single band.}
\label{ch5-fig03}
\end{figure}

The anti-tick, SB, LB, and construct \textbf{A}
lanes all show clear bands, 
but once again, the lane containing the
instruction strand shows multiple 
discrete bands.
This is due to the formation of aggregate hairpins -- 
multiple hairpins bound together.
This occurs when the hairpin stem first 
melts, then binds to the melted stem domain
of another hairpin, forming a 
double instruction, as illustrated below (see Figure~\ref{ch5-fig04}).
This can occur multiple times, and 
on the gel we see aggregates of up to six instructions forming in 
appreciable amounts. 

\begin{figure}[hbt]
\begin{center}
	\includegraphics[width=\textwidth]{ch5-fig04.eps}
\end{center}
\caption{ Hairpins can bind to other hairpins.}
\label{ch5-fig04}
\end{figure}


For this reason, we see multiple bands in the lane 
consisting of construct \textbf{B} in Figure~\ref{ch5-fig05}.
We know this is not due to any chain reaction, because 
no anti-tick had been added to that vessel.
On the other hand, when we 
look at the band in the \textbf{B} lane
representing only one instruction 
added (the band just above the one representing slight excess of 
intermediate \textbf{A}), we see there
is a band just above it in the lane 
containing \textbf{C}, indicating that the hybridization of a stoichiometric 
amount of anti-tick would not have caused a chain reaction in any case.

In order to confirm this, we decided to perform the experiment with an 
additional heat shock step to separate aggregate hairpins.
Before we added 
hairpins to any reaction vessel, we would
heat the hairpin strands to a high 
temperature.
This would open all hairpin stems and thereby separate all 
aggregate hairpins.
Then, we would quickly cool the strands on dry ice.
This 
quick cooling would favor intramolecular hybridization over intermolecular 
hybridization, ensuring that separate hairpins would not bind together.
We then hybridized these instructions 
to the growing constructs
with favorable results (see Figure~\ref{ch5-fig05}).

\begin{figure}[hbt]
\begin{center}
	\includegraphics{ch5-fig05.eps}
\end{center}
\caption{Hot-and-cold treatment eliminate hairpin aggregation, yielding
a clear dominant band in lane \textbf{B}. }
\label{ch5-fig05}
\end{figure}

The gel shows little
aggregation of instructions. 
In solution, there should be even less aggregation,
because the instruction strands 
used in the hybridizing reactions were used
immediately whereas the ones run in 
the gel had rested some time since the
heat shock.
The single clear 
band in the lane for \textbf{B} indicates the incorporation of a single 
instruction onto the base. 

The smearing observed around the band in lane \textbf{C} indicates some 
degree of chain reaction addition following the hybridization of anti-tick 
strands.
However, the smearing is minimal and the 
band density is clearly focused slightly above the band for \textbf{B}, 
indicating that the incorporation of a single
16bp anti-tick strand is the major 
product.

\subsection{Protocol Details}

Strands were designed by hand and ordered
from Integrated DNA Technologies. 
Strands were additionally purified by
running on a denaturing polyacrylamide 
gel in a normal TBE buffer at 55\r{ }C, followed by standard n-butanol 
extraction.

The small base and large base strands were suspended in TE buffer and 
combined in equal molar amounts.
The mixture was heated to 95\r{ }C for 20 
minutes and cooled incrementally (20 min
at 65\r{ }C, 20 min at 37\r{ }C, 20 
min at RT) before being stored at 4\r{ }C.

When adding anti-tick strands to the growing
complex, a number of strands 
approximately equal to the number of moles
of complex were measured out and 
the strands were dried using an evaporator.
The complex (in solution) was 
added to the dried strands and resuspended using a vortex. 

Instruction strands, prior to addition
and hybridizing to the growing complex, 
were heated at 95\r{ }C for approximately
5 minutes, and then rapidly cooled 
on dry ice until addition to disrupt any aggregate hairpins.
The 
instructions strands were incompletely
dried, as complete drying would allow 
for re-formation of hairpin aggregates upon resuspension.
The growing 
complex was added to the vial containing single instruction strands 
suspended in minimal 1x TE buffer and vortexed.


Hybridization for both anti-tick and instruction strands was accomplished by 
letting the strands sit at room temperature
for 30 minutes, then storing at 
14\r{ }C for at least 30 minutes.

At each hybridization step, a small aliquot of the intermediate product was 
removed and stored to be run on the native
polyacrylamide gel alongside the 
final product.
These changes in molar quantities of complexes were taken 
into account during subsequent strand additions.

\subsection{Experimental Conclusions/Future directions}

We have demonstrated that constructing
stacks of a specific length (and therefore clocked DNA programs) can
be achieved by a sequential addition 
process. 
We have also demonstrated 
that through simple stoichiometry, we
are able to limit the chain reactions 
that occur due to excess instructions and anti-tick strands during 
construction.
This may also have implications for stack deconstruction, where 
we seek to curb similar chain reactions with tick and tock strands.
We have 
also demonstrated the usefulness of heat
shock treatment of hairpin strands 
to ensure that aggregates are not incorporated into the stacks.

We have yet to determine how large we
can make these stacks and how reliably 
we can control chain reactions at larger sizes.
As they grow, native 
polyacrylamide gels may not be an informative
\index{gel}%
enough means of visualization, 
as they are not capable of resolving small differences in size beyond a 
certain point.
Fortunately, this is not a major concern because, for many
applications, fine-grained 
accuracy is not important. For example, in most parallel
processing applications, it will not matter if one stack
releases more than one copy of a given instruction strand. 
We must also determine how large the gated stacks can be. 

For the 
sake of simplicity, we may want to try using single-stranded instructions 
instead of hairpins.
Nevertheless, the experiments show that the principle 
of storing instructions within the DNA
in such a way that they can be released
through clock 
strands is entirely realizable.

\clearpage

