# Structural Dynamics of Peptiplexes Formed between Cationic Cell-Penetrating Peptides and DNA: A Comparative Study on TAT-HIV and NLS-SV40T

## TL;DR

This study compares how two cell-penetrating peptides interact with DNA, revealing differences in structure and efficiency for gene delivery.

## Contribution

The paper provides mechanistic insights into the structural dynamics of peptiplexes formed by TAT-HIV and NLS-SV40T with DNA.

## Key findings

- TAT-HIV/DNA peptiplexes show greater structural flexibility and self-assemble into clusters and nanofibrils.
- NLS-SV40T/DNA complexes form globule-studded nanoassemblies with internal 2D hexagonal columnar phases.
- NLS-SV40T/DNA peptiplexes are internalized more efficiently due to their compact structure and lower membrane damage.

## Abstract

Biomembranes evolved to protect cells and regulate exchange,
forming
a powerful barrier to large, charged macromolecules such as nucleic
acids. In recent years, this paradigm has been competently overturned
by soft biomaterials based on cell-penetrating peptides (CPPs). Herein,
we investigate and compare the structural dynamics of peptiplexes
formed between DNA and two cationic CPPs, TAT-HIV and NLS-SV40T. Combining
experimental approaches and molecular dynamics (MD) simulations, we
examined peptiplexes across mesoscopic scales to elucidate their supramolecular
assembly and correlate these features with cellular uptake. We found
that peptiplexes based on TAT-HIV exhibit greater structural flexibility,
adopting ordered secondary structures and self-assembling into clusters
and nanofibrils. In contrast, NLS-SV40T/DNA complexes retain random
coil configurations, forming globule-studded coiled nanoassemblies
with internal 2D hexagonal columnar phases. Calorimetry data indicated
that TAT-HIV/DNA complexation is more favorable and exothermic, whereas
NLS-SV40T binding to DNA is weaker and endothermic. MD simulations
supported the experiments by showing that NLS-SV40T moves across DNA
strands, settling into major grooves, whereas TAT-HIV bridges major
and minor grooves via persistent arginine-mediated H-bonds and stronger
energetics. Cell uptake assays showed that NLS-SV40T/DNA peptiplexes
are internalized comparatively more efficiently, likely due to their
more compact organization and lower lytic potential. Conversely, TAT-HIV
induces membrane damage, as observed by atomic force microscopy, suggesting
that its stronger electrostatics and enhanced H-bonding capacity may
contribute to lytic activity. The findings presented here bring mechanistic
insights into the structural landscape of peptiplexes, improving the
rationale that supports the design of peptide-mediated gene delivery
materials.