TY - JOUR

T1 - Deconvoluting the Effect of Cell-Penetrating Peptides for Enhanced and Controlled Insertion of Large-Scale DNA Nanopores

AU - Zhang, Xialin

AU - Malle, Mette Galsgaard

AU - Thomsen, Rasmus P.

AU - Sørensen, Rasmus Schøler

AU - Sørensen, Emily Winther

AU - Hatzakis, Nikos S.

AU - Kjems, Jørgen

N1 - Publisher Copyright: © 2024 The Authors. Published by American Chemical Society

PY - 2024

Y1 - 2024

N2 - DNA nanopores have emerged as powerful tools for molecular sensing, but the efficient insertion of large DNA nanopores into lipid membranes remains challenging. In this study, we investigate the potential of cell-penetrating peptides (CPPs), specifically SynB1 and GALA, to enhance the insertion efficiency of large DNA nanopores. We constructed SynB1- or GALA-functionalized DNA nanopores with an 11 nm inner diameter and visualized and quantified their membrane insertion using a TIRF microscopy-based single-liposome assay. The results demonstrated that incorporating an increasing number of SynB1 or GALA peptides into the DNA nanopore significantly enhanced the membrane perforation. Kinetic analysis revealed that the DNA nanopore scaffold played a role in prearranging the CPPs, which facilitated membrane interaction and pore formation. Notably, the use of pH-responsive GALA peptides allowed highly efficient and pH-controlled insertion of large DNA pores. Furthermore, single-channel recording elucidated that the insertion process of single GALA-modified nanopores into planar lipid bilayers was dynamic, likely forming transient large toroidal pores. Overall, our study highlights the potential of CPPs as insertion enhancers for DNA nanopores, which opens avenues for improved molecule sensing and the controlled release of cargo molecules.

AB - DNA nanopores have emerged as powerful tools for molecular sensing, but the efficient insertion of large DNA nanopores into lipid membranes remains challenging. In this study, we investigate the potential of cell-penetrating peptides (CPPs), specifically SynB1 and GALA, to enhance the insertion efficiency of large DNA nanopores. We constructed SynB1- or GALA-functionalized DNA nanopores with an 11 nm inner diameter and visualized and quantified their membrane insertion using a TIRF microscopy-based single-liposome assay. The results demonstrated that incorporating an increasing number of SynB1 or GALA peptides into the DNA nanopore significantly enhanced the membrane perforation. Kinetic analysis revealed that the DNA nanopore scaffold played a role in prearranging the CPPs, which facilitated membrane interaction and pore formation. Notably, the use of pH-responsive GALA peptides allowed highly efficient and pH-controlled insertion of large DNA pores. Furthermore, single-channel recording elucidated that the insertion process of single GALA-modified nanopores into planar lipid bilayers was dynamic, likely forming transient large toroidal pores. Overall, our study highlights the potential of CPPs as insertion enhancers for DNA nanopores, which opens avenues for improved molecule sensing and the controlled release of cargo molecules.

KW - bioconjugation

KW - cell-penetrating peptides

KW - DNA nanopore

KW - insertion enhancement

KW - single-liposome assay

KW - TIRF

U2 - 10.1021/acsami.3c18636

DO - 10.1021/acsami.3c18636

M3 - Journal article

C2 - 38573069

AN - SCOPUS:85189993215

VL - 16

SP - 18422−18433

JO - ACS applied materials & interfaces

JF - ACS applied materials & interfaces

SN - 1944-8244

IS - 15

ER -