User:Etienne Robillard/Notebook/Agent Ecoli:Plasmid cloning vectors for gene therapy with phosphohistidine based oligos

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= Plasmid cloning vectors for gene therapy with phosphohistidine-based oligos =
= Plasmid cloning vectors for gene therapy with phosphohistidine-based oligos =
__TOC__
__TOC__
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== Artificial DNA promoters: phosphoramidate-based oligonucleotide synthesis ==
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== Eukariotic DNA Promoters ==
cDNA:
cDNA:
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* amino-acid sequence signature (Myc epitope tag) = EQKLISEEDL
* amino-acid sequence signature (Myc epitope tag) = EQKLISEEDL
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Orthogonal DNA shell with Benzyl-derived (Bz) [http://en.wikipedia.org/wiki/Protecting_group#Carboxylic_acid_protecting_groups protecting groups]:
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=== Orthogonal DNA shell/therapy with Benzyl-derived phosphotriester protecting groups ===
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* note! note! Important: Can you elucidate the reasoning of using an carboxilic acid as protecting group for DNA assembly?  
* note! note! Important: Can you elucidate the reasoning of using an carboxilic acid as protecting group for DNA assembly?  
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** FIRST PROBABLE PROTECTION GROUP (A STRONG PROMOTER):
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*** 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.
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*** Note: Consensus promoter sequences are by definition strong promoters!
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** 2ND HIGHLY PROBABLE PROTECTION GROUP (A WEAK PROMOTER):
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*** 2-cyanoethyl : for protecting phosphoramidites based nucleosides?  XXX
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*** araBAD XXX
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*** phospho'''tri'''ester = a '''tri'''cyclic N-derived nitrogenous base bound to a Histidine residue made for '''DNA''' synthesis.
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**** 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.
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== References ==
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References:
 
* http://openwetware.org/images/1/1c/TheBacterialPromoter.pdf
* http://openwetware.org/images/1/1c/TheBacterialPromoter.pdf
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* http://en.wikipedia.org/wiki/Protecting_group#Carboxylic_acid_protecting_groups protecting groups
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== References ==
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* http://en.wikipedia.org/wiki/Oligonucleotide_synthesis#Nucleoside_phosphoramidites
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* http://en.wikibooks.org/wiki/Structural_Biochemistry/Nucleic_Acid/Nitrogenous_Bases/Purines

Revision as of 03:32, 15 November 2012

Plasmid cloning vectors for gene therapy with phosphohistidine-based oligos

Contents

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 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) 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 1
  • see also Oligonucleotide Synthesis
  • 1 mutation, 1 oligo assay (phosphoramidite-based oligo synthesis): [1]

M13K07 cloning in E. coli:

  • amino-acid sequence signature (Myc epitope tag) = EQKLISEEDL

Orthogonal DNA shell/therapy with Benzyl-derived phosphotriester protecting groups

  • note! note! Important: Can you elucidate the reasoning of using an carboxilic acid as protecting group for DNA assembly?
    • FIRST PROBABLE PROTECTION GROUP (A STRONG PROMOTER):
      • 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 (A WEAK PROMOTER):
      • 2-cyanoethyl : for protecting phosphoramidites based nucleosides? XXX
      • araBAD XXX
      • phosphotriester = a tricyclic 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.

References

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