DNA Oligonucleotide 3′-Phosphorylation by a DNA Enzyme

Alison J. Camden, Shannon M. Walsh, Sarah H. Suk, Scott K. Silverman

Research output: Contribution to journalArticle

Abstract

T4 polynucleotide kinase is widely used for 5′-phosphorylation of DNA and RNA oligonucleotide termini, but no natural protein enzyme is capable of 3′-phosphorylation. Here, we report the in vitro selection of deoxyribozymes (DNA enzymes) capable of DNA oligonucleotide 3′-phosphorylation, using a 5′-triphosphorylated RNA transcript (pppRNA) as the phosphoryl donor. The basis of selection was the capture, during each selection round, of the 3′-phosphorylated DNA substrate terminus by 2-methylimidazole activation of the 3′-phosphate (forming 3′-MeImp) and subsequent splint ligation with a 5′-amino DNA oligonucleotide. Competing and precedented DNA-catalyzed reactions were DNA phosphodiester hydrolysis or deglycosylation, each also leading to a 3′-phosphate but at a different nucleotide position within the DNA substrate. One oligonucleotide 3′-kinase deoxyribozyme, obtained from an N40 random pool and named 3′Kin1, can 3′-phosphorylate nearly any DNA oligonucleotide substrate for which the 3′-terminus has the sequence motif 5′-NKR-3′, where N denotes any oligonucleotide sequence, K = T or G, and R = A or G. These results establish the viabilty of in vitro selection for identifying DNA enzymes that 3′-phosphorylate DNA oligonucleotides.

Original languageEnglish (US)
Pages (from-to)2671-2676
Number of pages6
JournalBiochemistry
Volume55
Issue number18
DOIs
StatePublished - May 10 2016

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Phosphorylation
Oligonucleotides
DNA
Enzymes
Catalytic DNA
Substrates
Phosphates
Polynucleotide 5'-Hydroxyl-Kinase
RNA
Splints
Ligation
Hydrolysis
Phosphotransferases
Nucleotides
Chemical activation

ASJC Scopus subject areas

  • Biochemistry

Cite this

DNA Oligonucleotide 3′-Phosphorylation by a DNA Enzyme. / Camden, Alison J.; Walsh, Shannon M.; Suk, Sarah H.; Silverman, Scott K.

In: Biochemistry, Vol. 55, No. 18, 10.05.2016, p. 2671-2676.

Research output: Contribution to journalArticle

Camden, Alison J. ; Walsh, Shannon M. ; Suk, Sarah H. ; Silverman, Scott K. / DNA Oligonucleotide 3′-Phosphorylation by a DNA Enzyme. In: Biochemistry. 2016 ; Vol. 55, No. 18. pp. 2671-2676.
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