@@@@@@@@   This is a readme file for the retron design software used by the 2016 NTNU iGEM team. @@@@@@@@@

We are modeling the interaction of a deoxyribozyme RNA nuclease with a target RNA. We have two study cases:
In the first we are studying a self-inhibiting RBS, that gets activated by cleavage of the inhibiting arm at a 
specific locus:

mRNA:
5'-[RBS rev.compl.]--[random tract 1 (3)]--[recognition site no.1 (8)]--[cutting seq. (CGU)]--[recognition site no.2 (8)]--[random tract 2 (3)]--[RBS]--[original RFP mRNA]-3'

nucleases:
1:  5'-[high GC tail 1 (6)]--[recognition site no.2 (8) inv.compl.]--[nuclease catalytic site]--[recognition site no.1 (8) inv.compl.]--[high GC tail 2 (6)]-3'
2:  5'-[high GC tail 2 (6) inv.compl.]--[recognition site no.2 (8) inv.compl.]--[nuclease catalytic site]--[recognition site no.1 (8) inv.compl.]--[high GC tail 1 (6) inv.compl.]-3'

** (n) means n nucleotides long

In the second we are cleaving the mRNA of CI inhibitor.

mRNA:
usual CI mRNA

nucleases:
1:  5'-[high GC tail 1 (6)]--[recognition site no.2 (8) inv.compl.]--[nuclease catalytic site]--[recognition site no.1 (8) inv.compl.]--[high GC tail 2 (6)]-3'
2:  5'-[high GC tail 2 (6) inv.compl.]--[recognition site no.2 (8) inv.compl.]--[nuclease catalytic site]--[recognition site no.1 (8) inv.compl.]--[high GC tail 1 (6) inv.compl.]-3'


For the DNA and RNA folding we are using the ViennaRNA software package, more specifically RNAcofold. The current
version can either fold DNA with DNA or RNA with RNA. We therefore use an older version for the mRNA-nuclease interaction:
http://www.tbi.univie.ac.at/RNA/#other
For the interactions between two nucleases (DNA-DNA) we use the newest version of the software - 2.2.9:
http://www.tbi.univie.ac.at/RNA/#

We make 3 scripts: 
1) genRNA.sh - for the self-quenching RNA (input: 3)
2) genRNA_CI.sh - for the CI cleavage (input: 6 1.0)
3) mutagenize.sh - for fine-tuning the results of the two above (input: 10.0)

3 additional programs are there to assist the above scripts:
1) randseq.cpp   - generates random DNA or RNA sequences of defined length and GC-AT bias. (bias == 1 means equal amount of AT and GC, > 1 - more GC).
   example: ./randseq (RNA or R or DNA or D) (nucleotide number) (bias)
            ./randseq RNA 10 0.5
2) complement.cpp - generates the complementary sequence to a given input sequence 
   example: ./randseq (RNA or R or DNA or D) (sequence)
            ./randseq RNA augcaaccu
3) mut.cpp - makes n random mutations in the N first nucleotides from either end of a given DNA or RNA sequence and makes reverse complimentary mutations in a second DNA or RNA
   example: ./mut (RNA or R or DNA or D) (number of mutations n) (target bases from ends N) (sequence 1) (sequence 2)
            ./mut RNA 1 2 augcgga uccgcau

Additionally we provide the sequences we were using:
1) The RFP and CI mRNAs: RBS_1.in and RBS_11.in respectively.
2) The reverse complement of the RFP RBS: RBS_0.in

At first we run script 1 or 2 until we find a result where both nucleases are attached in the right place of the mRNA and have reasonable binding constants. 
We then use the mutagenize script in order to tune the binding constants of the two nucleases to become similar and large enough. 
Finally we use the newest version of the viennaRNA library to check that the nucleases attach to each other with much greater binding constant than to the mRNA.

### RUNNING THE SCRIPTS ###

To run these scripts, first compile the cpp files into executables with the same names. Then install
the needed viennaRNA libraries.

The scripts were run like this:
genRNA.sh 3
genRNA_CI.sh 6 1.0
mutagenize.sh 10.0

For further information please contact our chief designer. 

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