Difference between revisions of "User:Etienne Robillard/Notebook/Agent Ecoli:Plasmid cloning vectors for gene therapy with phosphohistidine based oligos"

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(Orthogonal DNA synthesis with Benzoyl-derived phosphotriester protecting groups (dA))
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= Plasmid cloning vectors for gene therapy with phosphohistidine-based oligos =
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== Eukariotic DNA Promoters ==
 
 
 
cDNA:
 
* N-6-benzoyl-deoxyadenosine phosphoramidite - Notice the P-N bound indicating the linkage of a phosphonate diester to a benzoyl atom :)
 
* N-3-oxo-hexanoyl-L-homoserine lactone - A light-inducible phosphohistidine-bound AHL ? A(denosine)TP/cAMP dependent.
 
* Reminder:
 
** NHC = (N heterocycle carbene) - Chemical compounds with a N chiral prodrug switch - highly enantioselective molecules
 
** N = indicates a Nitrogen atom
 
** P = a Phosphonate (inorganic state) - a compound for oligo synthesis using the [http://en.wikipedia.org/wiki/Oligonucleotide_synthesis#Nucleoside_phosphoramidites nucleoside phosphoramidite] method. 
 
** P-N = phosphoramidate (a Nitrogen atom bound to a Phosphonate ester in nucleophilic substitution).
 
 
 
deoxynucleotide synthesis:
 
* cDNA synthesis building block = deoxynucleoside + 3'-phosphonate = synthetic nucleotide analog (dATP) [http://dwb4.unl.edu/Teacher/NSF/C08/C08Links/www.dur.ac.uk/~dbl0www/Staff/Croy/cDNAfigs.htm 1]
 
 
 
oligodeoxyribonucleotide synthesis:
 
* short 50-200(?) bp restriction endonucleases enzymes for cDNA catalysis/plasmid vector cloning in ''E. coli''
 
* building blocks for oligonucleotides assembly with phosphoramidites method [http://en.wikipedia.org/wiki/Oligonucleotide 1]
 
* see also [http://openwetware.org/wiki/CH391L/S12/GeneandGenomeSynthesis#Oligonucleotide_Synthesis Oligonucleotide Synthesis]
 
* 1 mutation, 1 oligo assay (phosphoramidite-based oligo synthesis): [http://openwetware.org/wiki/Reviews/Directed_evolution/Library_construction#Directing_diversity:_Oligonucleotide-based_methods]
 
 
 
[http://openwetware.org/wiki/20.109(S07):Start-up_genome_engineering#Part_3:_Digest_M13K07 M13K07] cloning in ''E. coli'':
 
* amino-acid sequence signature (Myc epitope tag) = EQKLISEEDL
 
 
 
=== Orthogonal DNA synthesis with Benzoyl-derived phosphotriester protecting groups (dA) ===
 
 
 
* note! note! Important: Can you elucidate the reasoning of using an carboxilic acid as protecting group for DNA assembly?
 
** FIRST PROBABLE PROTECTION GROUP (dA):
 
*** Bz = Benzoyl (Bz, benzoic acid ester) : 2nd step in the catalysis of Benzilic acid from oxidation of Benzoic acid. See "benzoic acid". A very common [reagent] for site-directed oligonucleotide synthesis.
 
*** Note: Consensus promoter sequences are by definition strong promoters!
 
** 2ND HIGHLY PROBABLE PROTECTION GROUP (araBAD):
 
*** 2-cyanoethyl : for protecting phosphoramidites based nucleosides?  XXX
 
*** araBAD XXX
 
*** phospho'''tri'''ester = a '''tri'''cyclic N-derived nitrogenous base bound to a Histidine residue made for '''DNA''' synthesis.
 
* '''Thus phosphohistidine based phosphoramidates can be classified as purine analogues; the substance (Purines) was formally discovered by Emil Fischer during WW1 as a fundamental group of heterocyclic amino acids.'''
 
** see also [http://en.wikipedia.org/wiki/Piperidine Piperidine]
 
 
 
== References ==
 
 
 
* http://openwetware.org/images/1/1c/TheBacterialPromoter.pdf
 
* http://en.wikipedia.org/wiki/Protecting_group#Carboxylic_acid_protecting_groups protecting groups
 
* http://en.wikipedia.org/wiki/Oligonucleotide_synthesis#Nucleoside_phosphoramidites
 
* http://en.wikibooks.org/wiki/Structural_Biochemistry/Nucleic_Acid/Nitrogenous_Bases/Purines
 

Latest revision as of 05:06, 23 August 2015

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