Article Figures & Data
Figures
- Fig. 1.
Representative time to equilibrium of siRNA binding to total human plasma. Reference tip-subtracted sensorgrams depicting a titration of total human plasma interacting with biotinylated siRNA on streptavidin tips. Top plasma concentration is 3.3% (v/v) in buffer A followed by a 1:2 dilution series.
- Fig. 2.
(A) Workflow of determination of siRNA fu via ultrafiltration. Step 1: pretreat filter with detergent-containing buffer [we found that PBST (Table 1) and PBS+CHAPS (Supplemental Fig. 2) provided good recovery with a 50 kDa MWCO filter]. Step 2: add pre-equilibrated siRNA-spiked matrix into donor compartment of filter and centrifuge. Step 3: collect flow-through and quantify siRNA fu. (B) Depiction of siRNA-X Rh based on the crystal structure.
- Fig. 3.
PPB and liver protein binding of siRNA-X. (A) Cross-species comparison of fu,plasma across a range of therapeutically relevant siRNA concentrations. There was a significant increase in fu,plasma with concentration (P < 0.01) that was not dependent on species (determined by two-way ANOVA, GraphPad Prism). (B) siRNA-X fu,liver across a range of therapeutically relevant concentrations. Plasma measurements were performed in triplicate; liver measurements were performed in duplicate.
- Fig. 4.
Effect of chemical modifications on fu,plasma at 1 μM siRNA concentration. (A) Constructs tested with the sense strand depicted at the top (5′-3′) and the complementary antisense strand at the bottom. The RNA base sequence was constant across constructs, with only the conjugated ligand(s) (GalNAc and biotin), the ribose (2′-OMe, 2′-F), and the backbone (PS or phosphodiester) changing. (B) fu,plasma for each of the constructs in plasma; siRNA-X was also measured in serum. Results were compared using an ordinary one-way ANOVA with multiple comparisons in GraphPad Prism. Significant differences between the test article, siRNA-X in plasma, and the other constructs are reported as *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001.
- Fig. 5.
BLI sensorgrams of positive screening hits for select human plasma proteins binding to biotinylated siRNA-X ± GalNAc. A side-by-side comparison of anti-siRNA pAb (positive control), α-2-macroglobulin, α-thrombin, fibrinogen, and fibronectin binding to bn-siRNA-X with (left column) or without (right column) GalNAc conjugation. Titrations were 1:2 dilutions with a top concentration of 0.5 μM (except pAb = 0.2 μM).
- Fig. 6.
The relationship between Rh and MW for globular proteins does not hold for the dsRNA linear polymer according to Rh prediction based on dsRNA helical rise and the “siRNA rigid rod” assumption. siRNA is marked in red. GalNAc was not included in the Rh calculation. Linear regression was performed on the protein subset to estimation of the MW for a protein with an equivalent Rh to siRNA. The equation of the line obtained (with siRNA omitted) was y = 0.03509x + 1.233, and this was used to calculate the siRNA-protein MW equivalence value of 48 kDa (GraphPad Prism; calculations provided in Supplemental Calculations 2).
Tables
- TABLE 1
Comparison of percentage of siRNA-X recovery using different fu isolation techniques
fu Isolation Method MWCO % Recovery kDa Ultracentrifugation n/a 0.0062 ± 0.0003 Equilibrium dialysis 20 5.53 ± 7.13 Ultrafiltration 30 24.7 ± 1.40 Ultrafiltration 50 92.4 ± 11.6 - TABLE 2
Validation of 50 kDa MWMCO ultrafiltration method for determination of fu,plasma and fu,liver using well characterized small molecules
SM Matrix (Human) fu (Measured) fu (Reference) Reference Method Antipyrine Plasma 0.867 ± 0.110 0.9 Ultracentrifugation Timolol Plasma 0.347 ± 0.074 0.4 Ultracentrifugation Warfarin Plasma 0.043 ± 0.007 0.01a Ultracentrifugation Rosuvastatin Liver homogenate 0.14 ± 0.03 0.23b Equilibrium dialysis
Data Supplement
- Supplemental Data -
Supplemental methods and 6 figures.
- Supplemental Data -
In this issue
Plasma and Liver Protein Binding of GalNAc-Conjugated siRNA
