Burg J L, Juffras A M, Wu Y, Blomquist C L, Du Y
GENE-TRAK Corp, Framingham, MA 01701, USA.
Mol Cell Probes. 1996 Aug;10(4):257-71. doi: 10.1006/mcpr.1996.0035.
The addition of target-specific probe sequences within MDV RNA, an otherwise efficient template for Q beta replicase, generally resulted in RNA molecules with inferior replication properties, including reduced replication rates and poor sensitivities. We have discovered that the replication characteristics of MDV RNA molecules with internally placed probe sequences are dramatically affected by short RNA sequences (spacer elements) at the 5' and 3' ends of the probe sequence. For a given probe sequence, the sequences of the flanking spacer elements effected replication sensitivity by six orders of magnitude and replication rate by three fold. By taking advantage of spacer elements, internal MDV probes were developed that permitted the reproducible, real time, fluorescence detection of a single RNA molecule in less than 25 min through amplification with Q beta replicase. RNA structural analysis of such probes suggested that the spacer elements functioned by allowing the RNA to fold in a way which substantially maintained the tertiary structure of the MDV domain. MDV reporter probes with suitable replication properties were obtained from libraries of RNA molecules in which the probe sequence was flanked by many different spacer elements (generated by random nucleotide synthesis). We demonstrated that this is a general method for developing RNA reporter molecules which are rapidly and reproducibly amplified by Q beta replicase, even from a single molecule.
在MDV RNA（一种原本对Qβ复制酶而言高效的模板）中添加靶向特异性探针序列，通常会导致RNA分子的复制特性变差，包括复制速率降低和灵敏度不佳。我们发现，带有内部探针序列的MDV RNA分子的复制特性会受到探针序列5'和3'端短RNA序列（间隔元件）的显著影响。对于给定的探针序列，侧翼间隔元件的序列会使复制灵敏度相差六个数量级，复制速率相差三倍。通过利用间隔元件，开发出了内部MDV探针，通过Qβ复制酶扩增，可在不到25分钟的时间内对单个RNA分子进行可重复的实时荧光检测。对此类探针的RNA结构分析表明，间隔元件通过使RNA以基本维持MDV结构域三级结构的方式折叠来发挥作用。具有合适复制特性的MDV报告探针是从RNA分子文库中获得的，其中探针序列两侧有许多不同的间隔元件（通过随机核苷酸合成产生）。我们证明，这是一种开发RNA报告分子的通用方法，这些分子可被Qβ复制酶快速且可重复地扩增，即使是从单个分子开始。
