For the cohesive end ligation, a set of non-palindromic cohesive end sequences were utilized. This way, two asymmetric precursor ssDNA fragments (11a/11b) containing these cohesive ends at 5-termini would anneal into a stable asymmetric dumbbell-shaped duplex 11C (Scheme 1C). Both Exo I and III, the exonucleases used in this study, have 3-to 5-exonuclease ability. Exo I predominantly utilizes long protruding 3-ssDNA as substrates while Exo III digests short (< 4 nt) protruding 3'-overhangs, 3-termini at nicks, 3-blunt ends, and 3-recessed ends. As a result, overhangs longer than 4 nt at 3-termini are unlikely to be substrates for Exo I and III. Therefore, we initially utilized oligonucleotides having 14 nt long 5-overhangs (i.e., with 3-recessed termini) as the first set of precursors for cohesive end ligation synthesized cDNA. The structure of the final dumbbell-shaped cDNA, and stability of the stem region, along with secondary structure-free loop regions were verified using UNAFold (Fig. ). For investigating the role of cohesive end overhang length in NSA, we also utilized precursors having 9 and 5 nt 5-overhangs (12a/12b for making 12C, and 13a/13b for making 13C, respectively) (secondary structures shown in Figure and). The primer and HRCA primer sequences concerning the cDNA sequences and their binding sites in the cDNA secondary structures are also listed or illustrated in Table and Figure -S6, respectively. For investigating the DNA amplification concerning self-annealed and cohesive end ligation, a common primer 2 bearing sequence from DENV serotype 2 complementary to 1C, 11C, 12C, and 13C were used. For the HRCA studies on self-annealed and cohesive ligated cDNAs, HRCA primers 3 and 14 were utilized, respectively (Table ) . For the splint-padlock ligation, 9 as primer and 10 as HRCA primer was used (Table ) .