Molecular Logic Gates
Milan N. Stojanovic, Tiffany Elizabeth Mitchell, and Darko Stefanovic (2002) Deoxyribozyme-Based Logic Gates Journal of the American Chemical Society
Logic gates are a basic element in modern day computers, implementing simple mathematical operations in digital electronic circuits. Digital circuits operate in binary, distinguishing between two values: conventionally, "true" and "false", or "1" and "0". Within these circuits, logic gates perform binary operations on one or more inputs to produce a meaningful output. Common operations are the intuitively named "AND", "OR" and "NOT". These logical operations can be defined using truth tables (e.g. see the YES gate
below). In digital electronics, the binary values are represented by distinct "high" and "low" voltage signals, and logic gates are built using transistor switches. A good introduction to logic gates and digital circuits in computing can be found at wikipedia
. We have constructed molecular logic gates
using DNA as inputs, outputs and switches.
Molecular Logic Gate Components
The switch part of our molecular logic gate is derived from a deoxyribozyme, a nucleic acid enzyme that catalyzes DNA reactions. In this case the enzyme is a phosphodiesterase, which cleaves an oligonucleotide substrate (a short sequence of single-stranded DNA) into two shorter oligonucleotide products, or outputs.
DNA outputs and fluorescence monitoring
In order to monitor output formation, the outputs can be labeled with fluorescent dyes. In the case above, the Substrate is labeled with “red” channel TAMRA (T) dye, but its fluorescence is quenched by the Black-Hole 2 (BH2) quencher, which absorbs all of the TAMRA fluorescence. After cleavage, the TAMRA is separated from the BH2 and the fluorescence is no longer absorbed, leading to an increase in fluorescence within the mixture, which can be monitored via fluorescence spectroscopy. We have successfully used several combinations of fluorescent dye/quencher combinations, including Fluorescein/Black Hole Quencher 1 and Fluorescein/Tamra.
The fluorescence is merely a byproduct of the reaction for monitoring purposes. Output formation can be coupled to many different downstream events, such as the activation of a downstream gate, and release of a small molecule such as a drug.
DNA inputs and stem-loop controllers
The DNA enzyme is turned into a switch that is regulated by input DNA through the addition of specific stem-loop regions, which contain oligonucleotide binding regions. If input DNA (a short single-stranded oligonucleotide) is added, it will hybridize to the oligonucleotide binding region, causing the stem-loop to undergo a conformational change and break apart.
DNA inputs are highly selective, and will only hybridize to their specific complementary sequence. Thus it is possible to have many inputs and stem-loop regions in the same mixture without undesirable gate activation from input cross-reactivity. The use of stem-loop controlling structures also makes the gates fully modular, such that many oligonucleotide sequences can be placed for input binding in the loop regions.
Types of Logic gates
The simplest form of our molecular logic gates is the YES gate. In this gate, the DNA enzyme has been modified to include a stem-loop region that regulates the binding of substrate to the enzyme. If the stem-loop is closed, the substrate cannot bind, the enzyme is inactivated, and no outputs are formed. However, when input DNA is added, it hybridizes to the stem-loop region and alters the gate molecule's conformation. The conformational change causes the enzyme to become active, allowing cleavage of the substrate to produce the output DNA. i.e a YESx gate is active in the presence of a single input ix (see diagram below). The YES gate was first published as a catalytic molecular beacon
as an alternative method to detecting oligonucleotides through fluorescence.
Another type of molecular logic gate is the NOT gate. In this case, the catalytic core of the enzyme has been modified to include a stem-loop region that regulates enzyme activity. If the stem-loop is closed, the enzyme is active. However, when an input DNA is added, it hybridizes to the stem-loop region and alters the gate molecule's conformation. The conformational change causes the enzyme to become inactive, preventing cleavage of the substrate to produce output DNA. i.e a NOTz gate is active unless a single input iz is added, which inactivates the gate (see diagram below).
By using combinations of the YES and NOT loop structures, further Boolean logic gate structures can be made. The AND gate is made by combining two activating stem-loop structures:
The ANDNOT gate is made by combining an activating and an inhibitory stem-loop structure.
Combining two ANDNOT gates makes an XOR gate, which is active in the presence of either input (but not both). This gate was required for construction of our molecular half-adder.
The ANDANDNOT gate was first published in our first molecular automaton paper. This gate is created by combining two activating and one inhibitory stem-loop structures.
Significance of Logic gates
The logic gates shown above are the first chemical logic gates in which inputs and outputs are of the same kind (namely oligonucleotides). This allows the cascading of gates without any external interfaces. The inputs are compatible with sensor molecules
(aptamers) that could detect cellular disease markers, and the outputs can be tied to the release of small molecules, such as drugs. Thus it may eventually be possible to make therapeutic decisions cell-by-cell according to a complex decision function based on many attributes of the cell. This is sometimes referred to as "intelligent drug delivery".