JP2021016321A - インベージョン複合体の形成方法 - Google Patents
インベージョン複合体の形成方法 Download PDFInfo
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- JP2021016321A JP2021016321A JP2019132460A JP2019132460A JP2021016321A JP 2021016321 A JP2021016321 A JP 2021016321A JP 2019132460 A JP2019132460 A JP 2019132460A JP 2019132460 A JP2019132460 A JP 2019132460A JP 2021016321 A JP2021016321 A JP 2021016321A
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Landscapes
- Peptides Or Proteins (AREA)
Abstract
Description
(a)各々が互いに相補的な塩基配列を有し、
(b)各々、モノマー間がアミド結合で連結されてなり、且つ
(c)少なくとも一方の1本鎖人工核酸が、両者間のTm値を減少させる改変を含む、
2本の1本鎖人工核酸を、前記2本の1本鎖人工核酸に対して相補的な塩基配列を含む2本鎖核酸と接触させる工程を含む、インベージョン複合体の形成方法。
(a)各々が互いに相補的な塩基配列を有し、
(b)各々、モノマー間がアミド結合で連結されてなり、且つ
(c)少なくとも一方の1本鎖人工核酸が、両者間のTm値を減少させる改変を含む、
2本の1本鎖人工核酸。
本発明は、その一態様において、2本の1本鎖人工核酸であって、(a)各々が互いに相補的な塩基配列を有し、(b)各々、モノマー間がアミド結合で連結されてなり、且つ(c)少なくとも一方の1本鎖人工核酸が、両者間のTm値を減少させる改変を含む、2本の1本鎖人工核酸を、前記2本の1本鎖人工核酸に対して相補的な塩基配列を含む2本鎖核酸と接触させる工程を含む、インベージョン複合体の形成方法(本明細書において、「本発明の形成方法」と示すこともある。)に関する。以下に、これについて説明する。
で表される化合物が挙げられる。
本発明は、その一態様において、2本の1本鎖人工核酸であって、(a)各々が互いに相補的な塩基配列を有し、(b)各々、モノマー間がアミド結合で連結されてなり、且つ(c)少なくとも一方の1本鎖人工核酸が、両者間のTm値を減少させる改変を含む、2本の1本鎖人工核酸(本発明の核酸)、及び該2本の1本鎖人工核酸を含む組成物(本発明の組成物)に関する。
Fmoc固相合成法によるPNAの合成、精製を行った。合成したPNAを表1に示す。
合成したPNAを用いて119 bpのDNAに対してインベージョン実験を行った。119 bpのDNAはpBFP-N1のT817-A935に一致する配列である。合成した2本のPNAと2本鎖DNAとを混合し、ゲルおよびマイクロチップ電気泳動を行い、electrophoretic mobility shift assay(EMSA)によってインベージョン複合体の形成効率を評価した。
インベージョン試験1と同様にして試験した。EMSAの結果とインベージョン実験の反応条件とをあわせて、図6に示す。図6に示されるように、15mer及び12merのいずれのPNAを使用した場合も、PNA同士がparallel型になるように設計する方法(B)でインベージョンに成功した。
インベージョン試験1と同様にして試験した。EMSAの結果とインベージョン実験の反応条件とをあわせて、図7〜8に示す。図7〜8に示されるように、方法(A)及び方法(B)共に、インベージョン試験1と異なる配列を使用しても、インベージョンに成功した。
anti-parallel型及びparallel型のTm値を次のようにして測定した。終濃度がDNAおよびPNA: 1.0 μM、HEPES(pH 7.0): 5 mM、になるように、Milli-Qで調整し150 μLの溶液を調製した。温度可変型の紫外可視吸光光度計(JASCO)を用い、95 ℃→25 ℃、25 ℃→95 ℃の範囲で溶液の温度を変化させながら、260 nmの波長における吸光度を測定した。得られた温度変化に伴う吸光度の変化からTm値を算出した。
Claims (7)
- 2本の1本鎖人工核酸であって、
(a)各々が互いに相補的な塩基配列を有し、
(b)各々、モノマー間がアミド結合で連結されてなり、且つ
(c)少なくとも一方の1本鎖人工核酸が、両者間のTm値を減少させる改変を含む、
2本の1本鎖人工核酸を、前記2本の1本鎖人工核酸に対して相補的な塩基配列を含む2本鎖核酸と接触させる工程を含む、インベージョン複合体の形成方法。 - 前記2本の1本鎖人工核酸がパラレル型である、及び/又は前記2本の1本鎖人工核酸を相補関係に基づいて並べた場合に対向塩基が無い塩基が存在する、請求項1に記載の形成方法。
- 前記2本の1本鎖人工核酸がPNA(Peptide Nucleic Acid)である、請求項1又は2に記載の形成方法。
- 請求項1〜3のいずれかに記載の形成方法で得られるインベージョン複合体。
- 2本の1本鎖人工核酸であって、
(a)各々が互いに相補的な塩基配列を有し、
(b)各々、モノマー間がアミド結合で連結されてなり、且つ
(c)少なくとも一方の1本鎖人工核酸が、両者間のTm値を減少させる改変を含む、
2本の1本鎖人工核酸。 - 請求項5に記載の2本の1本鎖人工核酸を含む組成物。
- インベージョン複合体形成用である、請求項6に記載の組成物。
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Title |
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INAGAKI, MASAHITO ET AL., CHEMISTRY LETTER, vol. 48, no. 4, JPN6023011591, 5 April 2019 (2019-04-05), pages 341 - 344, ISSN: 0005086394 * |
RAY, ARGHYA AND NORDEN, BENGT, THE FASEB JOURNAL, vol. 14, no. 9, JPN6023011593, 1 June 2000 (2000-06-01), pages 1041 - 1060, ISSN: 0005086397 * |
SHIGI, NARUMI ET AL., MOLECULES, vol. Vol. 22, No. 10, Article No. 1586, JPN6023011592, 21 September 2017 (2017-09-21), pages 1 - 14, ISSN: 0005086396 * |
WITTUNG, PERNILLA ET AL., NATURE, vol. 368, JPN6023011589, 7 April 1994 (1994-04-07), pages 561 - 563, ISSN: 0005086395 * |
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