Scatter-Free UV–Visible Spectroscopy for Accurate and Precise RNA Quantification in Complex RNA Nanoparticle Formulations

TL;DR

This paper introduces a new UV-visible spectroscopy method for accurately measuring RNA in complex nanoparticle formulations without disrupting them.

Contribution

The novel scatter-free absorption spectroscopy (SFAS) method enables accurate RNA quantification in intact nanoparticles.

Findings

SFAS outperformed fluorescence-based methods in accuracy and precision across diverse RNA nanoparticle formulations.

SFAS was less affected by nanoparticle composition and measurement conditions compared to RiboGreen and SYTO 9.

The method is particularly effective for formulations resistant to disruption.

Abstract

Ribonucleic acid (RNA)-based drugs showed the potential
for treating
wide range of diseases. Their successful clinical use depends on developing
complex nanoparticle (NP) formulations made from diverse biomaterials.
Accurately quantifying total RNA concentration in complex formulations
is challenging, often requiring expensive, low-throughput methods
or fluorescence-based assays like RiboGreen that rely on effective
NP disruption. This study evaluates scatter-free absorption spectroscopy
(SFAS), a UV/Visible method that removes light scattering from NP
components and enables accurate total RNA quantification in intact
NPs. To validate SFAS, we employed diverse RNA formulations, including
lipid NPs, polymer and dendrimer hybrid lipid NPs, and cyclodextrin
nanocomplexes, which exhibit physicochemical characteristics that
can interfere with RNA quantification. Data obtained with SFAS were
compared to fluorescent-based assays utilizing RiboGreen and SYTO
9 dyes, which bind to RNA in free or encapsulated forms, respectively.
SFAS demonstrated superior accuracy, precision, and reproducibility
than fluorescence-based methods across all formulations, particularly
those showing resistance to disruption. RNA quantification by SFAS
was less influenced by NP composition and measurement conditions,
unlike the RiboGreen and SYTO 9. These findings demonstrate SFAS as
a versatile and reliable alternative to fluorescence-based assays
for accurate quantification of total RNA concentration in complex
RNA NP formulations.

Figures