We present a highly accurate method for identifying genes with conserved RNA secondary structure by searching multiple sequence alignments of a large set of candidate orthologs for correlated arrangements of reverse-complementary regions. This approach is growing increasingly feasible as the genomes of ever more organisms are sequenced. A program called MSARi implements this method and is significantly more accurate than existing methods in the context of automatically-generated alignments, making it particularly applicable to high-throughput scans. In our tests, it discerned ClustalW-generated multiple
sequence alignments of SRP or RNaseP orthologs from controls with 89.1% sensitivity at 97.5% specificity, and with 74.4% sensitivity with no false positives in 494 controls. We used MSARi to conduct the first comprehensive scan for secondary structure in mRNAs of coding genes, and found many genes with known mRNA secondary structure, in addition to finding compelling evidence for secondary structure in other genes. MSARi uses a novel method for coping with sequence redundancy that is likely to have applications in a large set of other comparison-based search methods. The program is available
for download from http://theory.csail.mit.edu/MSARi.