Yu Hua, Schwartz David C
Department of Chemistry, Laboratory for Molecular and Computational Genomics, University of Wisconsin-Madison, UW Biotechnology Center, 425 Henry Mall, Madison, WI 53706, USA.
Anal Biochem. 2008 Sep 1;380(1):111-21. doi: 10.1016/j.ab.2008.05.028. Epub 2008 May 24.
A surface-based approach is described for the transcriptional analysis of large, single DNA molecule templates and their imaged reaction products using RNA polymerase (RNAP). Results demonstrated that surfaces with a charge density supporting stretching of single DNA molecules to 70-80% of their full contour length were ideal for analysis of T7 RNAP transcription complexes on bound single template DNAs. Such DNA molecules were shown to sustain efficient transcription reactions and analysis, which enabled localization of transcription complexes on templates at kilobase resolution. Direct labeling of nascent RNA transcripts by the incorporation of a second fluorochrome into DNA templates promotes more robust and sensitive detection of punctates. Further characterization by RNase digestions, atomic force microscopy studies, and fluoro-immunolabeling revealed a "supercomplex" structure within a punctate where elongation complexes aggregate through entanglement of DNA and RNA strands from individual ternary elongation complexes. We have proposed mechanisms that underlie the supercomplex formation process. Whereas supercomplexes develop naturally in free solution, spatial constraints involved in a topologically limited system where template DNA is bound to the surface may facilitate the assembling process by stalling transcriptional elongation.
本文描述了一种基于表面的方法，用于对大型单DNA分子模板及其使用RNA聚合酶（RNAP）的成像反应产物进行转录分析。结果表明，电荷密度能将单DNA分子拉伸至其全长轮廓的70 - 80%的表面，非常适合分析结合在单模板DNA上的T7 RNAP转录复合物。此类DNA分子能够维持高效的转录反应和分析，从而能够以千碱基分辨率定位模板上的转录复合物。通过将第二种荧光染料掺入DNA模板对新生RNA转录本进行直接标记，可促进对斑点的更稳健和灵敏检测。通过核糖核酸酶消化、原子力显微镜研究和荧光免疫标记进一步表征发现，斑点内存在一种“超复合物”结构，其中延伸复合物通过来自单个三元延伸复合物的DNA和RNA链的缠结而聚集。我们提出了超复合物形成过程的潜在机制。虽然超复合物在自由溶液中自然形成，但在模板DNA结合到表面的拓扑受限系统中涉及的空间限制可能通过使转录延伸停滞来促进组装过程。
