Modifications of short RNAs at specific sites can be achieved commercially by solid-phase chemical synthesis method. However, labelling long RNAs is still challenging for the routine methods. Position-selective Labelling of RNA (PLOR) is a hybrid phase transcription method that allows to label RNAs at desired sites with great flexibility and decent efficiency. In principle, PLOR is a promising method for synthesis of long modified RNAs that are unable to be generated by solid-phase chemical synthesis and other methods. However, as a recently developed method, PLOR has been only applied to label a 71nt and a 104nt RNA, and the limited sequence applications of PLOR may hinder its potential usages. To extend PLOR to more RNAs, we tested the PLOR performances for various RNA sequences. Considering that the controlled transcriptional pauses at the initiation stage in PLOR may lead to different preferences on RNA sequences from in vitro transcription method, we here focused on identifying the effects of the 5ʹ-end and initiated lengths of RNA on PLOR. In addition, our work demonstrated that PLOR efficiencies also varied with linker sizes of DNA templates. This work can facilitate PLOR to be the choice of synthesizing long modified RNAs for more users in the near future.

可以通过固相化学合成方法在商业上实现对短RNA在特定位点的修饰。然而，对于常规方法而言，标记长RNA仍然具有挑战性。RNA的位置选择标记（PLOR）是一种杂种相转录方法，可以在所需位点以很大的灵活性和体面的效率标记RNA。原则上，PLOR是用于合成无法通过固相化学合成和其他方法生成的长修饰RNA的有前途的方法。然而，作为最近开发的方法，PLOR仅被用于标记71nt和104nt RNA，并且PLOR的有限的序列应用可能阻碍其潜在的用途。为了将PLOR扩展到更多RNA，我们测试了各种RNA序列的PLOR性能。在体外转录方法中，我们集中于鉴定RNA的5′末端和起始长度对PLOR的影响。此外，我们的工作表明PLOR效率也随DNA模板的接头大小而变化。这项工作可以促进PLOR在不久的将来成为更多用户合成长修饰RNA的选择。