Antisense small RNAs (abbreviated anti small RNA or anti-sRNA) are short RNA sequences (about 50-500 nucleotides long) that are complementary to other small RNA (sRNA) in the cell. sRNAs can repress translation via complementary base-pairing with their target mRNA sequence. Anti-sRNAs function by complementary pairing with sRNAs before the mRNA can be bound, thus freeing the mRNA and relieving translation inhibition. Anti-sRNAs lead to higher expression of mRNAs by inhibiting the action of sRNAs. Sponge RNA is another term used to describe anti-sRNAs. Discovery and Identification Methods While the mRNA-regulating small RNAs were discovered in 1984, the first natural anti-sRNA was only discovered in 2014 in an Escherichia coli model. The initial characterization of antisense small RNA within E. coli models were demonstrated through microarrays and computational predictions. Recent experiments have used Northern blot analysis and 5'-end mapping to correctly identify potential antisense sRNA candidates. RNA-Seq has emerged as a popular method for the identification of small RNA, since its ability to distinguish between messenger and structural RNA allows for increased sensitivity in sRNA analysis. Strand-specific RNA-Seq provides further characterization of sRNA by predicting transcript structures with enhanced accuracy. In 2019, a new algorithm called APERO was established which allows accurate genome-wide detection of small transcripts from paired-end bacterial RNA-Seq data. Paired-end bacterial sequencing allows for sequencing across both ends of the fragment, which increases the accuracy of the read by providing enhanced alignment. Protein-binding oriented techniques such as cross-linking immunoprecipitation, which isolates anti-sRNAs bound to proteins, have further contributed to the identification and detection of new anti-sRNA. A major contributor to this approach is the Hfq protein, a conserved RNA-binding protein that is known to attach various sRNAs. However, cross-linking immunoprecipitation fails to provide information on which two RNAs are interacting with each other, which is critical to identify the regulatory role of sRNAs. This shortcoming has been remedied by utilizing an RNA ligase to join the ends of the two RNAs that are interacting, allowing the mapping of sRNAs that are interacting with each other using RNA-Seq. Function Antisense small RNA are found in all domains of life, including Eukaryotes, Bacteria and Archaea. They are non-coding RNA sequences involved in regulatory processes, such as metabolism and aiding in transcription. Many anti-sRNAs are involved in regulatory activities to modulate gene expression, with the bulk of research exploring specific interactions within the bacterial domain. One example of this is established