Solid-phase synthesis, characterization and RNAi activity of branch and hyperbranch siRNAs

Maina, Anthony; Blackman, Brittany A.; Parronchi, Christopher J.; Morozko, Eva L.; Bender, Maria E.; Blake, Allan D.; Sabatino, David A.

doi:10.1016/j.bmcl.2013.08.033

Cited by 12 publications

(37 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The reaction produced a single product as detected by TLC and isolated following sample filtration and concentration in vacuo . The sample purity and identity of 5′‐levulinyl ribouridine, 2 , was confirmed by a combination of 1 H, 13 C NMR, and MS (Maina et al., ; Sabatino, ). The 5′‐levulinyl ribouridine, 2 , was then tritylated to the 5′‐levulinyl 2′ and 3′ –MMT ribouridine regioisomers, 3 and 4 , respectively, that were separable by column chromatography and characterized by NMR and MS. NMR COSY crosspeaks were used to identify the regioisomers by correlating the 2′H with the 2′OH for the 3′‐MMT isomer, 4 , and the 3′H with the 3′OH for the 2′MMT isomer, 3 .…”

Section: Resultsmentioning

confidence: 94%

“…The isolated 5′‐levulinyl 2′‐MMT ribouridine, 3 , was finally phosphitylated to the 3′‐phosphoramidite diastereomers, 5 . The isolated diastereomers, 5 , were confirmed by 31 P NMR and directly used as branchpoint synthon for the generation of V‐, Y‐ and >‐< shape siRNA (Maina et al., ; Sabatino, ).…”

Section: Resultsmentioning

confidence: 99%

“…The resultant V‐shape (2 siRNAs) and Y‐shape (3 siRNAs) were found to be more active than a linear siRNA control which silenced a single GRP78 mRNA site. The V‐ and Y‐shape siRNAs led to notable gene knockdown (50%–60%) and toxicity (5%–15%) in HepG2 liver cancer cells (Maina et al., ; Sabatino, ).…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Solid phase synthesis and self‐assembly of higher‐order siRNAs and their bioconjugates

et al. 2018

Self Cite

View full text Add to dashboard Cite

New methods for the synthesis of higher‐order siRNA motifs and their bioconjugates have recently gained widespread attention in the development of new and improved gene therapeutics. Our efforts aim to produce new chemical tools and protocols for the generation of modified siRNAs that screen for important oncogene targets as well as silence their activity for effective gene therapy in cancer models. More specifically, we have developed an efficient solution‐phase synthesis for the production of a ribouridine branchpoint synthon that can be effectively incorporated by solid phase synthesis within higher‐order RNA structures, including those adopting V‐, and Y‐ and >‐< shape RNA templates. Self‐assembly of complementary RNA to the template strands produced higher‐order siRNA nanostructures that were characterized by a combination of PAGE, DLS, and TEM techniques. In an effort to extend the repertoire of functionally diverse siRNAs, we have also developed solid phase bioconjugation strategies for incorporating bio‐active probes such as fatty acid appendages and fluorescent reporters. Taken together, these methods highlight the ability to generate higher‐order siRNAs and their bioconjugates for exploring the influence of modified siRNA structure on anti‐cancer activity.

show abstract

Section: Resultsmentioning

confidence: 94%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Solid phase synthesis and self‐assembly of higher‐order siRNAs and their bioconjugates

et al. 2018

Self Cite

View full text Add to dashboard Cite

show abstract

“…20,21 The linear and V- and Y-shaped RNA templates incorporating the antisense (A) or sense (S) single strands targeting GRP-75, 78, and 94 mRNA were synthesized by automated solid-phase RNA synthesis following our previously reported procedure. 11 In our synthetic approach, an orthogonally protected 5′-OLv 2′-OMMT ribouridine phosphoramidite was incorporated as branchpoint synthon to selectively and efficiently extend the linear RNA sequence into the V- and Y-shape RNA templates on solid phase. Following solid-phase synthesis, cleavage and deprotection, the crude RNA templates were purified by reverse-phase ion-pairing high-performance liquid chromatography (RP-IP-HPLC) in ≥96% purities, and their identities were confirmed by electrospray ionization mass spectrometry (ESI MS); see Table S1.…”

Section: Sirna Self-assemblymentioning

confidence: 99%

“…The RNA templates (namely, linear and V-, and Y-shaped RNA) were designed and synthesized according to our previously described methodology. 11 The V- and Y-shaped templates incorporate a branchpoint ribouridine (rU), which facilitates the hybridization of complementary sense (S) and antisense (A) RNA. These templates preorganizes the self-assembly of siRNA hybrid nanostructures having discrete sizes and shapes, including those belonging to circles, triangles, squares, rectangles, pentagons, hexagons, and poroustype structures.…”

Section: Figurementioning

confidence: 99%

RNAi Screening of the Glucose-Regulated Chaperones in Cancer with Self-Assembled siRNA Nanostructures

et al. 2016

Self Cite

View full text Add to dashboard Cite

The emerging field of RNA nanotechnology has been used to design well-programmed, self-assembled nanostructures for applications in chemistry, biology, and medicine. At the forefront of its utility in cancer is the unrestricted ability to self-assemble multiple siRNAs within a single nanostructure formulation for the RNAi screening of a wide range of oncogenes while potentiating the gene therapy of malignant tumors. In our RNAi nanotechnology approach, V- and Y-shape RNA templates were designed and constructed for the self-assembly of discrete, higher-ordered siRNA nanostructures targeting the oncogenic glucose regulated chaperones. The GRP78-targeting siRNAs self-assembled into genetically encoded spheres, triangles, squares, pentagons and hexagons of discrete sizes and shapes according to TEM imaging. Furthermore, gel electrophoresis, thermal denaturation, and CD spectroscopy validated the prerequisite siRNA hybrids for their RNAi application. In a 24 sample siRNA screen conducted within the AN3CA endometrial cancer cells known to overexpress oncogenic GRP78 activity, the self-assembled siRNAs targeting multiple sites of GRP78 expression demonstrated more potent and long-lasting anticancer activity relative to their linear controls. Extending the scope of our RNAi screening approach, the self-assembled siRNA hybrids (5 nM) targeting of GRP-75, 78, and 94 resulted in significant (50–95%) knockdown of the glucose regulated chaperones, which led to synergistic effects in tumor cell cycle arrest (50–80%) and death (50–60%) within endometrial (AN3CA), cervical (HeLa), and breast (MDA-MB-231) cancer cell lines. Interestingly, a nontumorigenic lung (MRC5) cell line displaying normal glucose regulated chaperone levels was found to tolerate siRNA treatment and demonstrated less toxicity (5–20%) relative to the cancer cells that were found to be addicted to glucose regulated chaperones. These remarkable self-assembled siRNA nanostructures may thus encompass a new class of potent siRNAs that may be useful in screening important oncogene targets while improving siRNA therapeutic efficacy and specificity in cancer.

show abstract

Synthesis, characterization, and biological activity of poly(arginine)-derived cancer-targeting peptides in HepG2 liver cancer cells

et al. 2014

Self Cite

View full text Add to dashboard Cite

The solid-phase synthesis, structural characterization, and biological evaluation of a small library of cancer-targeting peptides have been determined in HepG2 hepatoblastoma cells. These peptides are based on the highly specific Pep42 motif, which has been shown to target the glucose-regulated protein 78 receptors overexpressed and exclusively localized on the cell surface of tumors. In this study, Pep42 was designed to contain varying lengths (3-12) of poly(arginine) sequences to assess their influence on peptide structure and biology. Peptides were effectively synthesized by 9-fluorenylmethoxycarbonyl-based solid-phase peptide synthesis, in which the use of a poly(ethylene glycol) resin provided good yields (14-46%) and crude purities >95% as analyzed by liquid chromatography-mass spectrometry. Peptide structure and biophysical properties were investigated using circular dichroism spectroscopy. Interestingly, peptides displayed secondary structures that were contingent on solvent and length of the poly(arginine) sequences. Peptides exhibited helical and turn conformations, while retaining significant thermal stability. Structure-activity relationship studies conducted by flow cytometry and confocal microscopy revealed that the poly(arginine) derived Pep42 sequences maintained glucose-regulated protein 78 binding on HepG2 cells while exhibiting cell translocation activity that was contingent on the length of the poly(arginine) strand. In single dose (0.15 mM) and dose-response (0-1.5 mM) cell viability assays, peptides were found to be nontoxic in human HepG2 liver cancer cells, illustrating their potential as safe cancer-targeting delivery agents.

show abstract

Solid-phase synthesis, characterization and RNAi activity of branch and hyperbranch siRNAs

Cited by 12 publications

References 32 publications

Solid phase synthesis and self‐assembly of higher‐order siRNAs and their bioconjugates

Solid phase synthesis and self‐assembly of higher‐order siRNAs and their bioconjugates

RNAi Screening of the Glucose-Regulated Chaperones in Cancer with Self-Assembled siRNA Nanostructures

Synthesis, characterization, and biological activity of poly(arginine)-derived cancer-targeting peptides in HepG2 liver cancer cells

Contact Info

Product

Resources

About