TY - JOUR
T1 - Automated Synthesis and Purification of Guanidine-Backbone Oligonucleotides
AU - Skakuj, Kacper
AU - Bujold, Katherine E.
AU - Mirkin, Chad A.
N1 - Funding Information:
Research reported in this publication was supported by the National Cancer Institute of the National Institutes of Health under award U54CA199091. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The project was also supported by the Prostate Cancer Foundation and the Movember Foundation under award 17CHAL08, the Vannevar Bush Faculty Fellowship program sponsored by the Basic Research Office of the Assistant Secretary of Defense for Research and Engineering and funded by the Office of Naval Research through grant N00014-15-1-0043, and the NTU-NU Institute for NanoMedicine located at the International Institute for Nanotechnology, Northwestern University, USA, and the Nanyang Technological University, Singapore. K.E.B. gratefully acknowledges support from a Banting Fellowship from the Government of Canada. The authors would like to thank Robert Stawicki and Jennifer Delgado for their help in setting up the MerMadeTM 12 for DNG synthesis.
Funding Information:
Research reported in this publication was supported by the National Cancer Institute of the National Institutes of Health under award U54CA199091. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The project was also supported by the Prostate Cancer Foundation and the Movember Foundation under award 17CHAL08, the Vannevar Bush Faculty Fellowship program sponsored by the Basic Research Office of the Assistant Secretary of Defense for Research and Engineering and funded by the Office of Naval Research through grant N00014‐15‐1‐0043, and the NTU‐NU Institute for NanoMedicine located at the International Institute for Nanotechnology, Northwestern University, USA, and the Nanyang Technological University, Singapore. K.E.B. gratefully acknowledges support from a Banting Fellowship from the Government of Canada. The authors would like to thank Robert Stawicki and Jennifer Delgado for their help in setting up the MerMade 12 for DNG synthesis. TM
Publisher Copyright:
© 2020 Wiley Periodicals LLC
PY - 2020/6/1
Y1 - 2020/6/1
N2 - This protocol describes a method based on iodine and a base as mild coupling reagents to synthetize deoxyribonucleic guanidines (DNGs)—oligodeoxynucleotide analogues with a guanidine backbone. DNGs display unique properties, such as high cellular uptake with low toxicity and increased stability against nuclease degradation, but have been impeded in their development by the requirement for toxic and iterative manual synthesis protocols. The novel synthesis method reported here eliminates the need for the toxic mercuric chloride and pungent thiophenol that were critical to previous DNG synthesis methods and translates their synthesis to a MerMadeTM 12 automated oligonucleotide synthesizer. This method can be used to synthesize DNG strands up to 20 bases in length, along with 5′-DNG-DNA-3′ chimeras, at 1- to 5-μmol scales in a fully automated manner. We also present detailed and accessible instructions to adapt the MerMadeTM 12 oligonucleotide synthesizer to enable the parallel synthesis of DNG and DNA/RNA oligonucleotides. Because DNG linkages alter the overall charge of the oligonucleotides, we also describe purification strategies to generate oligonucleotides with varying lengths and numbers of DNGs, based on extraction or preparative-scale gel electrophoresis, along with methods to characterize the final products. Overall, this article provides an overview of the synthesis, purification, and handling of DNGs and mixed-charge DNG-DNA oligonucleotides.
AB - This protocol describes a method based on iodine and a base as mild coupling reagents to synthetize deoxyribonucleic guanidines (DNGs)—oligodeoxynucleotide analogues with a guanidine backbone. DNGs display unique properties, such as high cellular uptake with low toxicity and increased stability against nuclease degradation, but have been impeded in their development by the requirement for toxic and iterative manual synthesis protocols. The novel synthesis method reported here eliminates the need for the toxic mercuric chloride and pungent thiophenol that were critical to previous DNG synthesis methods and translates their synthesis to a MerMadeTM 12 automated oligonucleotide synthesizer. This method can be used to synthesize DNG strands up to 20 bases in length, along with 5′-DNG-DNA-3′ chimeras, at 1- to 5-μmol scales in a fully automated manner. We also present detailed and accessible instructions to adapt the MerMadeTM 12 oligonucleotide synthesizer to enable the parallel synthesis of DNG and DNA/RNA oligonucleotides. Because DNG linkages alter the overall charge of the oligonucleotides, we also describe purification strategies to generate oligonucleotides with varying lengths and numbers of DNGs, based on extraction or preparative-scale gel electrophoresis, along with methods to characterize the final products. Overall, this article provides an overview of the synthesis, purification, and handling of DNGs and mixed-charge DNG-DNA oligonucleotides.
KW - automated oligonucleotide synthesis
KW - deoxynucleic guanidines (DNG)
KW - oligonucleotide backbone modifications
KW - positively charged oligonucleotides
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U2 - 10.1002/cpnc.110
DO - 10.1002/cpnc.110
M3 - Article
C2 - 32530578
AN - SCOPUS:85086523380
VL - 81
JO - Current Protocols in Nucleic Acid Chemistry
JF - Current Protocols in Nucleic Acid Chemistry
SN - 1934-9270
IS - 1
M1 - e110
ER -