Chiến lược tự động gán tần số NMR cho RNA: ứng dụng cho K10 48 nucleotide

Journal of Biomolecular NMR - Tập 59 - Trang 231-240 - 2014
Barbara Krähenbühl1, Peter Lukavsky2, Gerhard Wider1
1Institute of Molecular Biology and Biophysics, ETH Zurich, Zurich, Switzerland
2CEITEC, Central European Institute of Technology, Masaryk University, Brno, Czech Republic

Tóm tắt

Bài báo trình bày một quy trình để gán tự động tần số NMR theo trình tự cho RNA được đánh dấu đồng nhất [13C, 15N]. Phương pháp này dựa trên một bộ bốn thí nghiệm quang phổ chiếu cao chiều tự động dựa trên liên kết và hai thí nghiệm qua không gian. Cách tiếp cận này được minh họa với một mẫu RNA stem-loop 0.3 mM có 48 nucleotide, K10, đóng vai trò trong việc định vị mRNA fs(1)K10 được trung gian bởi dynein của Drosophila. Phân tích tự động dữ liệu APSY dẫn đến danh sách đỉnh 3- đến 4-dimension rất chính xác và cụ thể. Chúng cung cấp một cơ sở đáng tin cậy cho việc gán tần số theo trình tự tiếp theo với thuật toán FLYA và đã dẫn đến việc gán tần số tự động hoàn toàn cho hơn 80% tần số của các phần tử 13C–1H tại các vị trí 1′, 2′, 5, 6 và 8 trong các nucleotide. Quy trình này cho thấy sự ổn định đối với nhiều đỉnh tạp chất, nồng độ thấp của RNA khá lớn này trong NMR, và các đặc điểm cấu trúc như vòng, bulges nucleotide đơn và các cặp base wobble không thuộc Watson–Crick. Hiện tại, không có thống kê hóa học dịch chuyển chính xác (như được sử dụng bởi FLYA) cho các vùng RNA lệch khỏi cấu trúc xoắn dạng A thông thường. Do đó, các danh sách đỉnh đáng tin cậy và chính xác là cần thiết cho gán tự động theo trình tự, như được cung cấp bởi APSY.

Từ khóa

#RNA #gán tần số NMR #phương pháp tự động #quang phổ chiếu cao chiều #K10

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