Substrate Specificity of T4 RNA Ligase 1 in Intramolecular Ligation of Single-Stranded DNA

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Assol R. Sakhabutdinova¹, Ayzilya A. Shakirova¹ and Ravil R. Garafutdinov^1,a*

¹Institute of Biochemistry and Genetics, Ufa Federal Research Center, Russian Academy of Sciences, 450054 Ufa, Bashkortostan, Russia

^* To whom correspondence should be addressed.

Received: November 10, 2025; Revised: June 18, 2026; Accepted: June 19, 2026

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Ligation of nucleic acids by DNA and RNA ligases is a routine method in molecular biology. Enzymatic ligation is increasingly used in the development of novel methods for detecting specific nucleotide sequences, as well as in non-biological DNA application. In this study, we investigated how the type of 5′- and 3′-terminal nucleotides affects the efficiency of single-stranded DNA cyclization. Model oligodeoxyribonucleotides (ODNs) 23, 39, and 45 nucleotides in length, representing all possible combinations of the donor and acceptor terminal nucleotides, were used as substrates. The substrate specificity of T4 RNA ligase 1 was assessed by analyzing the primary structure of amplicons generated through rolling circle amplification (RCA) of ligation products. For longer ODNs (39 and 45 nt), T4 RNA ligase 1 most efficiently cyclized substrates containing 5′-CC or 5′-TT donor dinucleotides and 3′-AA acceptor dinucleotide. Among the 23-nt ODNs, efficient cyclization was observed predominantly for a single substrate containing 5′-TT and 3′-AA terminal dinucleotides, likely reflecting a greater flexibility of AT-rich DNA. Substrates containing 5′-dGG and 3′-dGG dinucleotides showed the lowest propensity for cyclization. These findings provide guidance for selecting substrate sequences that maximize the yield of circular DNA products.

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KEY WORDS: RNA ligases, DNA cyclization, rolling circle amplification, nanopore sequencing, sequencing reads

DOI: 10.1134/S0006297925603983

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