Enzymatic synthesis of 2'-methylseleno-modified RNA

Siegmund, Vanessa; Santner, Tobias; Micura, Ronald; Marx, Andreas

Enzymatic synthesis of 2'-methylseleno-modified RNA

Dateien

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Datum

2011

Autor:innen

Siegmund, Vanessa

Santner, Tobias

Micura, Ronald

Marx, Andreas

Open Access-Veröffentlichung

Open Access Green

Sammlungen

Chemie

Publikationstyp

Zeitschriftenartikel

Publikationsstatus

Published

Erschienen in

Chemical Science. 2011, 2(11), pp. 2224-2231. ISSN 2041-6520. Available under: doi: 10.1039/C1SC00404B

Zusammenfassung

Selenium-derivatization of RNA is a powerful and advantageous alternative to conventional heavy atom derivatization techniques that are required for the phasing of X-ray crystallographic diffraction data. Among several possibilities, the 2'-methylseleno (2'-SeCH3) modification has been most widely explored and was responsible for a series of important RNA structure determinations, such as the Diels–Alder ribozyme or complexes of antibiotics to HIV dimerization initiation site (DIS) RNA. So far, 2'-SeCH3-RNA has only been accessible by chemical solid-phase synthesis for sizes of up to 50 nucleotides and up to about 100 nucleotides in combination with enzymatic ligation procedures. To overcome this limitation, here we present the enzymatic synthesis of 2'-SeCH3-RNA to open up access for the preparation of long selenium-modified RNA sequences, which cannot be accomplished by conventional chemical synthesis. Therefore, we first elaborated a synthetic route towards the 2'-methylseleno-2'-deoxyribonucleoside triphosphates of cytosine and uridine (2'-SeCH3–CTP and 2'-SeCH3–UTP). With these crucial derivatives in hand, we found that mutants of T7 RNA polymerase are able to incorporate 2'-SeCH3–CMP and 2'-SeCH3–UMP into RNA, while the wild-type polymerase fails to do so. This study demonstrates the efficient enzymatic synthesis of 2'-SeCH3-modified RNA and, thus, provides a thorough foundation for an alternative derivatization strategy in X-ray crystallographic structure analysis of larger RNAs. Such efforts are currently highly requested because of the steadily increasing number of novel non-coding RNAs whose structural features remain to be elucidated.

Fachgebiet (DDC)

540 Chemie

Zitieren

ISO 690

SIEGMUND, Vanessa, Tobias SANTNER, Ronald MICURA, Andreas MARX, 2011. Enzymatic synthesis of 2'-methylseleno-modified RNA. In: Chemical Science. 2011, 2(11), pp. 2224-2231. ISSN 2041-6520. Available under: doi: 10.1039/C1SC00404B

BibTex

@article{Siegmund2011Enzym-16383,
  year={2011},
  doi={10.1039/C1SC00404B},
  title={Enzymatic synthesis of 2'-methylseleno-modified RNA},
  number={11},
  volume={2},
  issn={2041-6520},
  journal={Chemical Science},
  pages={2224--2231},
  author={Siegmund, Vanessa and Santner, Tobias and Micura, Ronald and Marx, Andreas}
}

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    <dcterms:abstract xml:lang="eng">Selenium-derivatization of RNA is a powerful and advantageous alternative to conventional heavy atom derivatization techniques that are required for the phasing of X-ray crystallographic diffraction data. Among several possibilities, the 2'-methylseleno (2'-SeCH3) modification has been most widely explored and was responsible for a series of important RNA structure determinations, such as the Diels–Alder ribozyme or complexes of antibiotics to HIV dimerization initiation site (DIS) RNA. So far, 2'-SeCH3-RNA has only been accessible by chemical solid-phase synthesis for sizes of up to 50 nucleotides and up to about 100 nucleotides in combination with enzymatic ligation procedures. To overcome this limitation, here we present the enzymatic synthesis of 2'-SeCH3-RNA to open up access for the preparation of long selenium-modified RNA sequences, which cannot be accomplished by conventional chemical synthesis. Therefore, we first elaborated a synthetic route towards the 2'-methylseleno-2'-deoxyribonucleoside triphosphates of cytosine and uridine (2'-SeCH3–CTP and 2'-SeCH3–UTP). With these crucial derivatives in hand, we found that mutants of T7 RNA polymerase are able to incorporate 2'-SeCH3–CMP and 2'-SeCH3–UMP into RNA, while the wild-type polymerase fails to do so. This study demonstrates the efficient enzymatic synthesis of 2'-SeCH3-modified RNA and, thus, provides a thorough foundation for an alternative derivatization strategy in X-ray crystallographic structure analysis of larger RNAs. Such efforts are currently highly requested because of the steadily increasing number of novel non-coding RNAs whose structural features remain to be elucidated.</dcterms:abstract>
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