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<p>The construction of sequencing libraries mediated by Tn5 transposase (Tnp) has attracted growing interest from both industry and academia due to its considerable advantages over conventional ligation-based techniques. By consolidating DNA fragmentation and barcoding into a single process, Tn5 significantly reduces reaction time and improves barcoding efficiency. Nonetheless, the widespread application of this enzyme is constrained by insertion bias, which constitutes a major limitation. We employed a comprehensive approach that amalgamated in silico and in vivo methodologies to examine this issue. Initially, docking simulations were conducted to model the binding interactions between the Tnp dimer and double-stranded DNA, comparing specific and nonspecific sequences. Subsequent molecular dynamics simulations provided insights into distinct binding mechanisms. In the Tnp-specific DNA complex, the residue of Q118 was observed to penetrate the major groove, forming bidentate hydrogen bonds with the guanine (G) and thymine (T) bases, a feature essential for specific molecular recognition. Guided by these insights, we performed saturation mutagenesis at the Q118 position, thereby experimentally confirming its role in DNA specificity. This investigation led to the identification of an “enhanced variant” (Q118T), which demonstrated a reduced insertion bias. Collectively, our findings provide molecular-level insights into the recognition of Tnp-DNA, highlighting the pivotal role of hydrogen bonding in determining specificity. This study advances the mechanistic understanding of Tn5 Tnp and has the potential to contribute to the development of improved tools for library construction.</p>

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Rational modification of Tn5 transposase insertion bias via in silico modeling and in vivo assay

  • 期刊论文

  • 发表日期：

    2026年1月26日

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  • Zhong Ni,
  • Huayun Jia,
  • Huimin Zhou,
  • Fangyuan Song,
  • Dongfeng Chen,
  • Lili Shen,
  • Huayou Chen

摘要

The construction of sequencing libraries mediated by Tn5 transposase (Tnp) has attracted growing interest from both industry and academia due to its considerable advantages over conventional ligation-based techniques. By consolidating DNA fragmentation and barcoding into a single process, Tn5 significantly reduces reaction time and improves barcoding efficiency. Nonetheless, the widespread application of this enzyme is constrained by insertion bias, which constitutes a major limitation. We employed a comprehensive approach that amalgamated in silico and in vivo methodologies to examine this issue. Initially, docking simulations were conducted to model the binding interactions between the Tnp dimer and double-stranded DNA, comparing specific and nonspecific sequences. Subsequent molecular dynamics simulations provided insights into distinct binding mechanisms. In the Tnp-specific DNA complex, the residue of Q118 was observed to penetrate the major groove, forming bidentate hydrogen bonds with the guanine (G) and thymine (T) bases, a feature essential for specific molecular recognition. Guided by these insights, we performed saturation mutagenesis at the Q118 position, thereby experimentally confirming its role in DNA specificity. This investigation led to the identification of an “enhanced variant” (Q118T), which demonstrated a reduced insertion bias. Collectively, our findings provide molecular-level insights into the recognition of Tnp-DNA, highlighting the pivotal role of hydrogen bonding in determining specificity. This study advances the mechanistic understanding of Tn5 Tnp and has the potential to contribute to the development of improved tools for library construction.