Bahr Ute, Aygün Hüseyin, Karas Michael
Cluster of Excellence Macromolecular Complexes, Institute of Pharmaceutical Chemistry, University of Frankfurt, 60438 Frankfurt, Germany.
Anal Chem. 2008 Aug 15;80(16):6280-5. doi: 10.1021/ac800605z. Epub 2008 Jul 11.
While MALDI-MS is widely accepted for quality control of synthetic oligonucleotides, this method has been regarded as not applicable for a control of the purity and correct annealing of double strands. The results presented here show that the double-strand intensities measured by MALDI-MS maintain and reflect the solution conditions. Using a single-stranded RNA as internal standard, the double-strand intensity can be determined by measuring the intensity ratio of the single strands to the standard under "native" conditions and after denaturation with formic acid. For siRNAs with fully matched 20-21 base pairs, relative intensities of the double strands are between 94 and 97.2%. The stability determined by MALDI-MS for different RNA duplexes correlates well with calculated T m values and the content of G-C pairs. Furthermore, the quantification method enables one to determine an excess of one single strand and the contribution of duplex formation by truncated strands. The results show that MALDI-MS is a fast and reliable method for quality control of synthetic siRNA.
虽然基质辅助激光解吸电离质谱法（MALDI-MS）在合成寡核苷酸的质量控制方面已被广泛接受，但该方法一直被认为不适用于双链体纯度和正确退火的控制。此处呈现的结果表明，通过MALDI-MS测量的双链强度能够维持并反映溶液条件。以单链RNA作为内标，双链强度可通过在“天然”条件下以及用甲酸变性后测量单链与标准的强度比来确定。对于具有完全匹配的20 - 21个碱基对的小干扰RNA（siRNA），双链的相对强度在94%至97.2%之间。通过MALDI-MS测定的不同RNA双链体的稳定性与计算得到的解链温度（Tm）值以及鸟嘌呤 - 胞嘧啶（G - C）对的含量密切相关。此外，该定量方法能够确定一条单链的过量以及截短链对双链形成的贡献。结果表明，MALDI-MS是一种用于合成siRNA质量控制的快速且可靠的方法。
