While the inability of Exo III in digesting protruding 3-termini longer than 4 nt has been well characterized (52), we could not find literature reports concerning the length-dependent digestion efficiency of 3-recessive ends by Exo III. We hypothesized a possible mechanism on how a shorter cohesive end length may contribute to greater NSA. Earlier, during probing conditions C1 -C4, we observed and postulated relatively poor Exo III substrate selectivity for cohesive end precursor 11a/11b. It has led to a relatively lower difference for NSA between C1 and C2 conditions in 11C compared to that for self-annealing and splint-padlock ligated cDNAs 1C and 4C, respectively. In addition to a poor substrate binding, the cDNA affinity of Exo III might as well be inversely correlated with the 5protruding cohesive end length through a yet undiscovered mechanism. As a result, these unligated precursors having 5, 9, and 14 nt long 5-protruding cohesive ends are being partially digested with the worst to best affinity and digestion efficiency, respectively (Scheme S2). Due to this, the effective postdigestion concentration of cohesive end precursors would be the highest for 5 nt and lowest for 14 nt. These unligated, poorly digested precursors, present along with ligated cDNA in the same solution, would be forming a strand-displacement complex with the cDNA (Scheme S2). The efficiency of generation of such strand displacement complex would be correlating to the concentrations of 5, 9, 14 nt precursors, i.e., highest and lowest for 5 and 14 nt precursors, respectively. The strand displacement complex, through the 3-exonuclease activity of 29 polymerase, would be a direct substrate of the later . As a result, the NSA, resulting from 3-exonuclease activity of 29 polymerase, would follow a descending trend for 5, 9, and 14 nt cohesive end precursors. While this unproven hypothesis successfully explains the observed trends in length-dependent NSA, it was not pursued any further for mechanistic validation in this work and will be taken up in future investigations. As explained earlier, precursor oligonucleotides having long (> 4 nt) 3-protruding overhangs are poor substrates for Exo III and therefore not utilized in this study for cohesive end ligation .