How to make a pseudoknot

[JR1]

STEP ONE:

LET’S CREATE A SEED SEQUENCE:

Genome - NCBI - NLM
select a genome of a interesting bug from the list below
get a list
select one from list
select download
select (ALL(2) and Genome sequences (FASTA)
select download @ bottom of that window
find that downloaded file = ncbi_dataset.zip
open the zip file
dig down into the data file looking for a file that starts with GCK
with extension .fna
change the .fna to .txt
open that .txt file
You are seeing the genome of that bug
globally change all "T"s to "U"s
There you go - thousands of seeds split into 100 long sequence
that you can now stomp into the lab puzzle all day long.

STEP TWO:

Create the puzzle

Stomp any sequence into the puzzle
use the thunder bolt/sequence stomper - position 35 is good
the results configuration is interesting so i will proceed with this one
notice the DOT PLOT on the RHS of the screen is very dirty
and no pseudoknot
let’s fix that now

STEP THREE

Clean up

freeze the puzzle and remove everything except for the stacks
strengthen the stacks with AU’ and GCs removing GUs
put Cs or whatever to stop all the blinking AAAAs so it’s not so annoying
look at the DOT PLOT
you can now see your 3 stacks without a lot of dirty background
but still no pseudoknot

STEP FOUR

make your pseudoknot

mentally pick where you want your pseudoknot to be
I decided to remove the fist stack - gone
I have chosen the hairpin and the loop segment to the right of the hairpin
put a strong but not too strong sequence into the hairpin
if your hairpin sequence conflicts with the stacks - too strong
now start the complementary loop sequence NT by NT until the pseudoknot is strong enough to
show up on the screen
now look at the DOT PLOT - the pseudoknot shows up faintly as a third stack.
Oh boy - we are headed in the right direction.
I think I want to make the pseudoknot stand out more - not so recessive

STEP FIVE

the balancing act

at this point in the design process you may want to put on your sidewalk scientist hat.
and think about what you want to do
my goal for this puzzle is to make the pseudoknot show up stronger in the DOT PLOT
and I can accomplish the task by making the pseudoknot stronger by adding one on more complementary NT to the loop sequence, to the hairpin sequence or reducing the strength of the stacks by switching GCs to AUs, reversing GCs, or making AU’s to GC’s to GU’s.
lots of ways to go
the darker the DOT PLOT sequence the strong the sequence is
try to make the stacks and pseudoknots around the same shade of black
post when you are satisfied with the DOT PLOT structures shade
post several versions - commenting in the comment section what you did to compare results easily months from now - use you imagination - have fun
if additional mysteriously show up you show get rid of them or incorporate them into the puzzle - they will skew results