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Open Dataset XSDM4ADX


Correlating the Structure and Gene Silencing Activity of Oligonucleotide-Loaded Lipid Nanoparticles Using Small-Angle X-Ray Scattering

Abstract

Individual ASO-LNP samples and the screening sample plates were prepared by a robotic liquid handler-assisted, high-throughput solvent-injection method that we developed previously. Briefly, a liquid handler (TECAN EVO, NC, USA) was used to dispense the ASO dissolved in a citrate buffer (25 mM, pH 4) into a 96-well sample plate, as well as to mix individual lipid stocks at certain molar ratios to generate different lipid mixtures. Formulated ASO-LNPs composed of 40 mol% MC3, 2 mol% DMG-PEG-2k, 10 mol% DSPC, and 48 mol% cholesterol at an N/P ratio of 2. Next we tested how modifying formulation parameters, including the PEG-lipid concentration and N/P ratio, affected these SAXS parameters. We varied the PEG-lipid concentration from 2 to 5% and N/P ratios of ASO-LNPs from 2 to 5. We next examined the potential of LNP morphology changes after purification. During the purification process, the pre-purified LNPs are buffer exchanged into a physiologically relevant phosphate-buffered saline (PBS) buffer with pH 7.4.

Experimental description

SAXS data were collected in the high throughput mode (HT-SAXS) using the Advanced Light Source SIBYLS beamline 12.3.1 at the Lawrence Berkeley National Laboratory (CA, USA). X-ray wavelength was set at λ = 1.216 Å, and the sample-to-detector distance was 2070 mm, resulting in a scattering vector, q, ranging from 0.01 Å-1 to 0.45 Å-1. The scattering vector is defined as q = 4πsinθ/λ, where 2θ is the scattering angle. Experiments were performed at 20 °C as described elsewhere 29. Briefly, the sample was exposed for 10 s with the detector framing at 0.3 s to maximize the signal while merging the SAXS signal using the SAXS FrameSlice application (https://bl1231.als.lbl.gov/ran). No radiation damage was observed during the 10 s exposure, and all collected frames were merged.

File description

HTS1_pre_purification.dat = SAXS curve of ASO-LNP composed of 40 mol% MC3, 2 mol% DMG-PEG-2k, 10 mol% DSPC, and 48 mol% cholesterol at an N/P ratio of 2 in a citrate buffer (25 mM, pH 4) HTS2_pre_purification.dat = SAXS curve of ASO-LNP composed of 40 mol% MC3, 5 mol% DMG-PEG-2k, 10 mol% DSPC, and 45 mol% cholesterol at an N/P ratio of 2 in a citrate buffer (25 mM, pH 4). HTS3_pre_purification.dat = SAXS curve of ASO-LNP composed of 40 mol% MC3, 2 mol% DMG-PEG-2k, 10 mol% DSPC, and 48 mol% cholesterol at an N/P ratio of 5 in a citrate buffer (25 mM, pH 4). HTS1_post_purification.dat = SAXS curve of ASO-LNP composed of 40 mol% MC3, 2 mol% DMG-PEG-2k, 10 mol% DSPC, and 48 mol% cholesterol at an N/P ratio of 2 in PBS buffer HTS2_post_purification.dat = SAXS curve of ASO-LNP composed of 40 mol% MC3, 5 mol% DMG-PEG-2k, 10 mol% DSPC, and 45 mol% cholesterol at an N/P ratio of 2 in a PBS buffer. HTS3_post_purification.dat = SAXS curve of ASO-LNP composed of 40 mol% MC3, 2 mol% DMG-PEG-2k, 10 mol% DSPC, and 48 mol% cholesterol at an N/P ratio of 5 in a PBS buffer.

Created

2023-02-07

Published

2024-08-08

Data collection technique

HT-SAXS

Journal DOI

Source

Beamline

Wavelength

1.216 Å

Sample to Detector Distance

2.07 m


Submitting Author

Michal Hammel

Lawrence Berkeley National Laboratory, The SIBYLS Beamline

United States of America

[email protected]

Collaborators

Project Leader

Michal Hammel

[email protected]


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Complete Set of SAS Data Files

The complete SAS dataset is downloadable as a zip file.

  • LNP-ASO_pre_post_purification.zip Download


Individual SAS Data Files (total 0)

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Supplemental Data and Supporting Materials (total 0)

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SAXS Similarity SAXS FrameSlice


Sample:

  • Macromolecule 1: ASO-LNP
    • Sample Full Name: ASO-LNP
    • Sample Type: Other
    • Source Organism:
    • Source Organism NCBI Taxonomy ID:
    • Expression System:
    • Expression NCBI Taxonomy ID:
    • Uniprot ID's:
    • Sequence or Chemical Formula: