# Team:MIT/ResultsProcessing

(Difference between revisions)
 Revision as of 19:43, 26 October 2012 (view source)Eerik (Talk | contribs)← Older edit Revision as of 22:59, 26 October 2012 (view source)Eerik (Talk | contribs) Newer edit → Line 14: Line 14:

Overview

Overview

- Processing information involves using various logic gates to compute the value of a logic expression. Every such expression can be evaluated using a combination of AND, OR and NOT logic gates, making these three gates crucial. Qian et al. already designed and demonstrated AND and OR gates, however did not build a modular NOT gate. + Processing information involves using various logic gates to compute the value of a logic expression. Every such expression can be evaluated using a combination of AND, OR and NOT logic gates, making these three gates crucial for any information processing unit. Qian et al. already designed and demonstrated AND and OR gates, however did not build a modular NOT gate.

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The NOT gate (signal inverter) design we eventually used comprises of multiple double- and single-stranded species. The most important of these are NOT gate (A), output (B) and buffer (C) complexes.
The NOT gate (signal inverter) design we eventually used comprises of multiple double- and single-stranded species. The most important of these are NOT gate (A), output (B) and buffer (C) complexes.
- There are two important cases to consider: first the case where there is no input present (in digital terms, the input is 0), and second the case where there is a saturating level of input (digitally represented as a 1). We aim to convert the 0 into a 1 and the 1 into a 0 - the basic operation of a NOT gate. Refer to the diagram below throughout the explanation. + There are two important cases to consider: first the case where there is no input present (in digital terms, the input is 0), and second the case where there is a saturating level of input (digitally represented as a 1). We aim to convert the '0' input into a '1' output and the '1' into a '0' - the basic operation of a NOT gate. Refer to the diagram below throughout the explanation.

Case 1: No input present -> high signal
Case 1: No input present -> high signal
- Here, the output strand (B) reversibly reacts with the NOT gate (A). The reaction is reversible because the output strand only displaces the green section on the NOT gate, but not further, meaning that the reaction could go back as well. This reversible reaction allows the output strand to react to a downstream buffer and amplifier to produce a high signal (a new nucleic acid strand is released and now active). + Here, the output strand (B) reversibly reacts with the NOT gate (A). The reaction is reversible because the output strand only displaces the green section on the NOT gate, but not further, meaning that the reaction could go back as well. This reversible reaction allows the output strand to react to a downstream buffer (C) and amplifier (D) to produce a high signal (a new active nucleic acid strand is released).

Case 2: Input present -> low signal
Case 2: Input present -> low signal
- Here, the input strand and output strand both react with the NOT gate. The input strand displaces up to the pink region and the output strand displaces up to the green region. This is irreversible, as once strand displacement happens from both sides, the complex separates into two double-stranded species and there is no toehold to initiate the reverse reaction. Now the output strand is stuck and cannot be buffered and amplified to produce a signal (no new nucleic acid strand is released).

+ Here, the input strand and output strand both react with the NOT gate. The input strand displaces up to the pink region and the output strand displaces up to the green region. This is irreversible, as once strand displacement happens from both sides, the complex separates into two double-stranded species. There is no toehold to initiate the reverse reaction, so the output strand is "stuck". The output therefore cannot be buffered or amplified to produce a signal: no new active nucleic acid strand is released.

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Motivation

Motivation

- Producing RNA strand-displacement circuits inside cells is non-trivial, as the gate:output complexes in these circuits need to be properly base-paired as soon as they are produced. Otherwise, a loose output strand might trigger a downstream gate:output or simply anneal with an incorrect gate. This problem can be removed by producing gate:output complexes as single transcripts that then fold to form a stem-loop type structure. This poses a second problem, since this affects the kinetics of reactions involving an input signal and the gate:output as this reaction now changes from a tri-molecular reaction to a bi-molecular reaction. To solve this problem, we incorporate Hammerheads in the gate:output transcript. These same arguments and solutions apply for the threshold complex as well. + Producing RNA strand-displacement circuits inside cells is non-trivial, as the gate:output complexes in these circuits need to be properly base-paired as soon as they are produced. Otherwise, a loose output strand might trigger a downstream gate:output or simply anneal with an incorrect gate. This problem can be removed by producing gate:output complexes as single transcripts that then fold to form a stem-loop type structure. This poses a second problem, since this in turn affects the kinetics of reactions involving an input signal and the gate:output, as this reaction now changes from a tri-molecular reaction (involving the input, gate and output strands) to a bi-molecular reaction (now the gate:output complex is just one strand). To solve this problem, we incorporate Hammerheads in the gate:output transcript to cut the gate:output transcript into two.
+ These same arguments and solutions apply for the threshold complex (introduced by Qian et al.) as well.

- The Hammerhead ribozyme is a self-cutting ribozyme: upon being transcribed to RNA, it folds into a particular secondary structure and catalyzes its own cleavage. + The Hammerhead ribozyme is a self-cutting ribozyme: upon being transcribed to RNA, it folds into a particular secondary structure which enables it to catalyze its own cleavage.

## Revision as of 22:59, 26 October 2012

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# Overview

Processing information involves using various logic gates to compute the value of a logic expression. Every such expression can be evaluated using a combination of AND, OR and NOT logic gates, making these three gates crucial for any information processing unit. Qian et al. already designed and demonstrated AND and OR gates, however did not build a modular NOT gate.

We demonstrated the first NOT gate compatible with toehold-mediated strand displacement in an in vitro setting using DNA. This NOT gate is modular in vivo thanks to production and degradation of RNA circuits.

We also introduced the Hammerhead ribozyme, a powerful self-cleaving RNA structural motif, to iGEM and the parts registry. This ribozyme can be used to produce the double-stranded RNA complexes required for processing in vivo.

# Our NOT Gate Design, Simulations and Optimization

## Design

The NOT gate (signal inverter) design we eventually used comprises of multiple double- and single-stranded species. The most important of these are NOT gate (A), output (B) and buffer (C) complexes.
There are two important cases to consider: first the case where there is no input present (in digital terms, the input is 0), and second the case where there is a saturating level of input (digitally represented as a 1). We aim to convert the '0' input into a '1' output and the '1' into a '0' - the basic operation of a NOT gate. Refer to the diagram below throughout the explanation.

Case 1: No input present -> high signal
Here, the output strand (B) reversibly reacts with the NOT gate (A). The reaction is reversible because the output strand only displaces the green section on the NOT gate, but not further, meaning that the reaction could go back as well. This reversible reaction allows the output strand to react to a downstream buffer (C) and amplifier (D) to produce a high signal (a new active nucleic acid strand is released).

Case 2: Input present -> low signal
Here, the input strand and output strand both react with the NOT gate. The input strand displaces up to the pink region and the output strand displaces up to the green region. This is irreversible, as once strand displacement happens from both sides, the complex separates into two double-stranded species. There is no toehold to initiate the reverse reaction, so the output strand is "stuck". The output therefore cannot be buffered or amplified to produce a signal: no new active nucleic acid strand is released.

Diagram of our NOT gate. See the paragraph above for a more detailed description.

# In Vitro Results for Our NOT Gate

To test the functionality of the NOT gate as expressed by its transfer function (relating the input levels to output signal strengths) we performed in vitro studies with DNA as the nucleic acid for strand displacement. The output signal strand was fed to a reporter complex. This reporter, when triggered by a signal, will fluoresce in the red channel. Our experiments involved measuring these fluorescence levels for various amounts of initial input levels.

# Hammerhead Ribozymes for Producing RNA Circuits In Vivo

## Motivation

Producing RNA strand-displacement circuits inside cells is non-trivial, as the gate:output complexes in these circuits need to be properly base-paired as soon as they are produced. Otherwise, a loose output strand might trigger a downstream gate:output or simply anneal with an incorrect gate. This problem can be removed by producing gate:output complexes as single transcripts that then fold to form a stem-loop type structure. This poses a second problem, since this in turn affects the kinetics of reactions involving an input signal and the gate:output, as this reaction now changes from a tri-molecular reaction (involving the input, gate and output strands) to a bi-molecular reaction (now the gate:output complex is just one strand). To solve this problem, we incorporate Hammerheads in the gate:output transcript to cut the gate:output transcript into two.
These same arguments and solutions apply for the threshold complex (introduced by Qian et al.) as well.

The Hammerhead ribozyme is a self-cutting ribozyme: upon being transcribed to RNA, it folds into a particular secondary structure which enables it to catalyze its own cleavage.

Software rendering of the minimum free energy structure of the N117 Hammerhead ribozyme. Left: abstract ball-and-chain representation, right: 3D rendering of RNA. The left side of each diagram represents the 5' end of the ribozymes. The N117 Hammerhead sequence was derived from Yen et al. (2004).

## Design

Hammerheads may allow us to transcribe gate:output double-stranded complexes in vivo in a single transcript. The DNA coding for the gate:output complex would include the sequences for each strand, separated by sequences coding for the Hammerhead RNA. When this DNA is transcribed into RNA, the gate and output sequences bind by Watson-Crick base-pairing, while the Hammerhead folds and cleaves within its sequence, resulting in the correct double-stranded gate:output two-strand complex. See below for the schematic of this process.

Using Hammerheads to make gate:output complexes in vivo. See the above paragraph for a detailed description.