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Abstract

The most widely used technique for the production of DNA aptamers/oligonucleotides is chemical synthesis. Despite its effectiveness, this technique cannot be performed “in house”, making the user fully dependent on a supplier. In this work, we present a simplified method by which it is possible to enzymatically produce DNA aptamers “in house”. This new method uses the rolling circle replication followed by a unique cleavage step using the SchI endonuclease. Potentially, any oligonucleotide can be produced by the enzymatic method proposed in this study. To illustrate, we present the production of three variations of the 31-TBA aptamer, a single stranded DNA which has anticoagulant action.

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References

Gragoudas

, Adamis

, Cunningham

, Feinsod

and Guyer

. (2004). Pegaptanib for neovascular age-related macular degeneration. N Engl J Med, 351:2805–2816.

Keefe

, Pai

and Ellington

. (2010). Aptamers as therapeutics. Nat Rev Drug Discov, 9:537–550.

Suh

and Jaykus

. (2013). Nucleic acid aptamers for capture and detection of Listeria spp. J Biotechnol, 167:454–461.

Lohmann

, Stougaard

and Koch

. (2007). A new enzymatic route for production of long 5′-phosphorylated oligonucleotides using suicide cassettes and rolling circle DNA synthesis. BMC Biotechnol, 7:49.

Umekage

and Kikuchi

. (2009). In vitro and in vivo production and purification of circular RNA aptamer. J Biotechnol, 139:265–272.

Wang

, Gou

and Xu

S-Y

. (2010). Polymerase-endonuclease amplification reaction (PEAR) for large-scale enzymatic production of antisense oligonucleotides. PLoS One, 5:e8430.

Ducani

, Kaul

, Moche

, Shih

and Högberg

. (2013). Enzymatic production of ‘monoclonal stoichiometric’ single-stranded DNA oligonucleotides. Nat Methods, 10:647–652.

Nelissen

FHT

, Goossens

EPM

, Tessari

and Heus

. (2015). Enzymatic preparation of multimilligram amounts of pure single-stranded DNA samples for material and analytical sciences. Anal Biochem, 475:68–73.

Kikin

, Zappala

, D'Antonio

and Bagga

. (2008). GRSDB2 and GRS_UTRdb: databases of Quadruplex forming G-rich sequences in pre-mRNAs and mRNAs. Nucleic Acids Res, 36:D141–D148.

10.

Lorenz

, Bernhart

, zu Siederdissen

, Tafer

, Flamm

, Stadler

and Hofacker

. (2011). ViennaRNA Package 2.0. Algorithms Mol Biol, 6:26.

11.

Mathews

, Disney

, Childs

, Schroeder

, Zuker

and Turner

. (2004). Incorporating chemical modification constraints into a dynamic programming algorithm for prediction of RNA secondary structure. Proc Natl Acad Sci U S A, 101:7287–7292.

12.

Ikebukuro

, Okumura

, Sumikura

and Karube

. (2005). A novel method of screening thrombin-inhibiting DNA aptamers using an evolution-mimicking algorithm. Nucleic Acids Res, 33:e108.

13.

Zavyalova

, Golovin

, Reshetnikov

, Mudrik

, Panteleyev

, Pavlova

and Kopylov

. (2011). Novel modular DNA aptamer for human thrombin with high anticoagulant activity. Curr Med Chem, 18:3343–3350.

14.

Bock

, Griffin

, Latham

, Vermaas

and Toole

. (1992). Selection of single-stranded DNA molecules that bind and inhibit human thrombin. Nature, 355:564–566.

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“In-House” Production of Single Stranded Oligodeoxyribonucleotides

Abstract

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Supplementary Material