Lan Tao, Putta Mallikarjuna Reddy, Wang Daqing, Dai Meiru, Yu Dong, Kandimalla Ekambar R, Agrawal Sudhir
Idera Pharmaceuticals, Inc., 167 Sidney Street, Cambridge, MA 02139, USA.
Biochem Biophys Res Commun. 2009 Aug 28;386(3):443-8. doi: 10.1016/j.bbrc.2009.06.036. Epub 2009 Jun 11.
Single-stranded RNAs act as ligands of Toll-like receptors (TLRs) 7 and 8 and induce immune responses. In the present study, we have designed and synthesized phosphorothioate oligoribonucleotides (ORNs) with self-complementary sequences that form duplex structures with either 3'- or 5'-overhanging sequences. We studied the new ORNs for their duplex formation, nuclease stability, and ability to induce immune-stimulatory activate through TLR7 and TLR8 in TLR-transfected cell lines, human PBMCs, human pDCs, and in vivo in mice. Thermal melting and gel electrophoresis studies showed that all ORNs formed secondary structures and that the thermal stability of the duplex is depended on the length and GC composition of the duplex. Nuclease stability of ORNs increased with increasing thermal stability of the duplex formed. All ORN showed TLR8 activity in HEK293 cells, and induced cytokine and chemokine production in human PBMC cultures. In addition to TLR8 activity, two ORNs containing a 'CUGAAUU' motif in the duplex-forming region induced immune stimulation through TLR7 in HEK293 cells, human PBMC and pDC cultures, and in vivo in mice. These results suggest that secondary structures in ORN provide nuclease stability and lead to stimulation of immune responses through TLR8 as well as TLR7 depending on the presence of specific nucleotide motifs.
单链RNA作为Toll样受体（TLR）7和8的配体，可诱导免疫反应。在本研究中，我们设计并合成了具有自互补序列的硫代磷酸寡核糖核苷酸（ORN），这些序列可形成具有3'或5'突出序列的双链结构。我们研究了新型ORN在转染TLR的细胞系、人外周血单核细胞（PBMC）、人浆细胞样树突状细胞（pDC）以及小鼠体内形成双链的情况、核酸酶稳定性以及通过TLR7和TLR8诱导免疫刺激激活的能力。热变性和凝胶电泳研究表明，所有ORN均形成二级结构，且双链的热稳定性取决于双链的长度和GC组成。ORN的核酸酶稳定性随着所形成双链热稳定性的增加而提高。所有ORN在HEK293细胞中均表现出TLR8活性，并在人PBMC培养物中诱导细胞因子和趋化因子的产生。除了TLR8活性外，在双链形成区域含有“CUGAAUU”基序的两种ORN在HEK293细胞、人PBMC和pDC培养物以及小鼠体内通过TLR7诱导免疫刺激。这些结果表明，ORN中的二级结构提供了核酸酶稳定性，并根据特定核苷酸基序的存在通过TLR8以及TLR7刺激免疫反应。
