In this study, splint-padlock ligation was chosen due to its representation in the highest number of reported RCA-mediated bioanalytical applications (Figure ). Since NSA could originate from undigested precursor oligonucleotides, self-annealed ligation seemed a rational choice as it uses only one precursor oligonucleotide for an intramolecular-templated ligation. Unlike CircLigase-assisted ligation which also utilizes a single precursor oligonucleotide, self-annealed ligation is not restricted by any enzymatic sequence selectivity . Cohesive end ligation was selected (over similar bluntended ligation that produces symmetrical cDNA) due to its ability to incorporate sequence diversity and synthesize asymmetric long cDNAs. The strategy of cDNA synthesis by self-annealed, splintpadlock, and cohesive end mediated ligation involved the design of sequences (Table ), and simulation of the secondary structures (Figures ), enzymatic 5-phosphorylation, and ligation (Scheme 1). For the preparation of a dumbbell-shaped cDNA 1C with self-annealed ligation, the stem regions of the precursor oligonucleotide 1 must fold into a stable duplex to bring 5-PO4 and 3-OH termini in proximity (Scheme 1A). Furthermore, the loop region sequences were carefully chosen to be predominantly free of any secondary structure. The presence of secondary structures in the loop region of 1C and folding stability of the stem region as duplex were checked using UNAFold (IDT) simulation (Figure ) . For splint-padlock mediated ligation (Scheme 1B), a 38 nt long splint 5 bearing sequence from Escherichia coli malB gene and a precursor padlock oligonucleotide sequence 4 was chosen to prepare cDNA 4C (Figure ). Additionally, the role of splint length was investigated using shorter splints (30, 24, and 16 nt, oligonucleotides 6 -8, respectively) without changing the final cDNA sequence 4C itself or the ligation site (Figure ).