S1). After UV-cross-linking, the membrane was prehybridized in PerfectHyb plus hybridization buffer
(Sigma, St Louis, MO) at 65 °C. A biotin-labeled antisense oligonucleotide (5′-GTGTGTTCCCTTGCGTCCCA-3′) probe was then added directly to the prehybridization buffer and incubated overnight at 37 °C. After hybridization, the membrane was washed twice with 0.1× SSC/0.1% SDS at room temperature. The signals were detected by using the chemiluminescent nucleic acid detection module (Thermo Scientific) according to the manufacturer’s protocol. Small size cDNA libraries of S. mutans were analysed by deep sequencing, which gave 19 million sequence reads. The sequences composed of 15–26 nt were extracted as valid sRNAs and were compared with Doxorubicin price buy Pexidartinib various RNA databases (NCBI and Rfam). The length distribution of all sRNAs (mappable reads) is shown in Fig. 1. sRNAs and their extended sequences (flanking sequences) were analysed for hairpin structure prediction and classification. Of these sequenced sRNAs, 17.6% (3 372 405 reads) and 6.5% (1 239 481 reads) were mapped to ribosomal RNAs (and others) and mRNAs, respectively (Table 1). Others belonged to the group of RNAs that were not
blasted to any reference RNA databases and therefore may represent the fraction of novel RNAs. sRNAs were considered as putative msRNAs if they are able to form hairpins with flanking nucleotide sequences in the genome. msRNAs with more than 100 clone counts are detailed in Table 2. Seven selected msRNAs were verified by qRT-PCR
using specific TaqMan probe and primer sets (Fig. 2). This analysis revealed a rough correlation between the number of msRNAs, identified Resminostat by the deep sequencing, and their cellular content. Six of seven tested candidates may form complementary duplexes with other msRNAs registered in this study (Fig. 2b). In animals, during typical miRNA biogenesis, one strand of an RNA duplex is preferentially selected for combining with a silencing complex, whereas the other one, known as the miRNA* strand, is inactivated or degraded (O’Toole et al., 2006). However, some miRNA* sequences were reported as guide miRNAs with abundant expression (Okamura et al., 2008; Jagadeeswaran et al., 2010). Revealing putative msRNA* sequences for certain msRNAs (Fig. 2b and Table 2), however, we were unable to verify msRNA* expression by qRT-PCR because the software failed to design specific TaqMan probe and primer sets, which may be due to their RNA structure or small size (Table 2). Although the validated msRNA-428 can also form a short hairpin structure with its extended sequence, the corresponding msRNA* was not found among the registered reads. msRNA-428 is encoded by the genomic region located in front of 16S rRNA genes (one or two mismatches with S. mutans UA159 genomic DNA). The cellular form of msRNA-428 was tested by Northern blotting (Fig. 2c), which revealed a single band of the expected size (20 nt).