Sliding over the blocks in enzyme-free RNA copying – one-pot primer extension in ice

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

Resumé

Template-directed polymerization of RNA in the absence of enzymes is the basis for an information transfer in the 'RNA-world' hypothesis and in novel nucleic acid based technology. Previous investigations established that only cytidine rich strands are efficient templates in bulk aqueous solutions while a few specific sequences completely block the extension of hybridized primers. We show that a eutectic water/ice system can support Pb 2+/Mg 2+-ion catalyzed extension of a primer across such sequences, i.e. AA, AU and AG, in a one-pot synthesis. Using mixtures of imidazole activated nucleotide 5′-monophosphates, the two first "blocking" residues could be passed during template-directed polymerization, i.e., formation of triply extended products containing a high fraction of faithful copies was demonstrated. Across the AG sequence, a mismatch sequence was formed in similar amounts to the correct product due to U·G wobble pairing. Thus, the template-directed extension occurs both across pyrimidine and purine rich sequences and insertions of pyrimidines did not inhibit the subsequent insertions. Products were mainly formed with 2′-5′-phosphodiester linkages, however, the abundance of 3′-5′-linkages was higher than previously reported for pyrimidine insertions. When enzyme-free, template-directed RNA polymerization is performed in a eutectic water ice environment, various intrinsic reaction limitations observed in bulk solution can then be overcome.

OriginalsprogEngelsk
Artikelnummere75617
TidsskriftP L o S One
Vol/bind8
Udgave nummer9
Sider (fra-til)e75617
ISSN1932-6203
DOI
StatusUdgivet - sep. 2013

Fingeraftryk

Copying
pyrimidines
Ice
Polymerization
polymerization
ice
RNA
Eutectics
linkage (genetics)
Enzymes
enzymes
cytidine
transfer RNA
Cytidine
Pyrimidines
imidazoles
Water
information exchange
Insertional Mutagenesis
purines

Citer dette

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abstract = "Template-directed polymerization of RNA in the absence of enzymes is the basis for an information transfer in the 'RNA-world' hypothesis and in novel nucleic acid based technology. Previous investigations established that only cytidine rich strands are efficient templates in bulk aqueous solutions while a few specific sequences completely block the extension of hybridized primers. We show that a eutectic water/ice system can support Pb 2+/Mg 2+-ion catalyzed extension of a primer across such sequences, i.e. AA, AU and AG, in a one-pot synthesis. Using mixtures of imidazole activated nucleotide 5′-monophosphates, the two first {"}blocking{"} residues could be passed during template-directed polymerization, i.e., formation of triply extended products containing a high fraction of faithful copies was demonstrated. Across the AG sequence, a mismatch sequence was formed in similar amounts to the correct product due to U·G wobble pairing. Thus, the template-directed extension occurs both across pyrimidine and purine rich sequences and insertions of pyrimidines did not inhibit the subsequent insertions. Products were mainly formed with 2′-5′-phosphodiester linkages, however, the abundance of 3′-5′-linkages was higher than previously reported for pyrimidine insertions. When enzyme-free, template-directed RNA polymerization is performed in a eutectic water ice environment, various intrinsic reaction limitations observed in bulk solution can then be overcome.",
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Sliding over the blocks in enzyme-free RNA copying – one-pot primer extension in ice. / Löffler, Philipp M. G.; Groen, Joost; Dörr, Mark; Monnard, Pierre-Alain.

I: P L o S One, Bind 8, Nr. 9, e75617, 09.2013, s. e75617.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - Sliding over the blocks in enzyme-free RNA copying – one-pot primer extension in ice

AU - Löffler, Philipp M. G.

AU - Groen, Joost

AU - Dörr, Mark

AU - Monnard, Pierre-Alain

PY - 2013/9

Y1 - 2013/9

N2 - Template-directed polymerization of RNA in the absence of enzymes is the basis for an information transfer in the 'RNA-world' hypothesis and in novel nucleic acid based technology. Previous investigations established that only cytidine rich strands are efficient templates in bulk aqueous solutions while a few specific sequences completely block the extension of hybridized primers. We show that a eutectic water/ice system can support Pb 2+/Mg 2+-ion catalyzed extension of a primer across such sequences, i.e. AA, AU and AG, in a one-pot synthesis. Using mixtures of imidazole activated nucleotide 5′-monophosphates, the two first "blocking" residues could be passed during template-directed polymerization, i.e., formation of triply extended products containing a high fraction of faithful copies was demonstrated. Across the AG sequence, a mismatch sequence was formed in similar amounts to the correct product due to U·G wobble pairing. Thus, the template-directed extension occurs both across pyrimidine and purine rich sequences and insertions of pyrimidines did not inhibit the subsequent insertions. Products were mainly formed with 2′-5′-phosphodiester linkages, however, the abundance of 3′-5′-linkages was higher than previously reported for pyrimidine insertions. When enzyme-free, template-directed RNA polymerization is performed in a eutectic water ice environment, various intrinsic reaction limitations observed in bulk solution can then be overcome.

AB - Template-directed polymerization of RNA in the absence of enzymes is the basis for an information transfer in the 'RNA-world' hypothesis and in novel nucleic acid based technology. Previous investigations established that only cytidine rich strands are efficient templates in bulk aqueous solutions while a few specific sequences completely block the extension of hybridized primers. We show that a eutectic water/ice system can support Pb 2+/Mg 2+-ion catalyzed extension of a primer across such sequences, i.e. AA, AU and AG, in a one-pot synthesis. Using mixtures of imidazole activated nucleotide 5′-monophosphates, the two first "blocking" residues could be passed during template-directed polymerization, i.e., formation of triply extended products containing a high fraction of faithful copies was demonstrated. Across the AG sequence, a mismatch sequence was formed in similar amounts to the correct product due to U·G wobble pairing. Thus, the template-directed extension occurs both across pyrimidine and purine rich sequences and insertions of pyrimidines did not inhibit the subsequent insertions. Products were mainly formed with 2′-5′-phosphodiester linkages, however, the abundance of 3′-5′-linkages was higher than previously reported for pyrimidine insertions. When enzyme-free, template-directed RNA polymerization is performed in a eutectic water ice environment, various intrinsic reaction limitations observed in bulk solution can then be overcome.

KW - DNA Primers/chemistry

KW - Ice

KW - Imidazoles/chemistry

KW - RNA/chemical synthesis

KW - Ribonucleotides/chemistry

U2 - 10.1371/journal.pone.0075617

DO - 10.1371/journal.pone.0075617

M3 - Journal article

C2 - 24058695

VL - 8

SP - e75617

JO - P L o S One

JF - P L o S One

SN - 1932-6203

IS - 9

M1 - e75617

ER -