High Affinity and Specific Binding of HIV-1 TAR RNA by a Tat-Derived Oligourea

High Affinity and Specific Binding of HIV-1 TAR RNA by a Tat-Derived Oligoure

HIV-1 TAR RNA Recognition by an Unnatural Biopolymer

HIV-1 TAR RNA Recognition by an Unnatural Biopolyme

A New Strategy for Site-Specific Protein Modification: Analysis of a Tat Peptide−TAR RNA Interaction

Site-specific modification of proteins and peptides with reporter molecules provides a powerful research tool in chemistry and biology. We report the synthesis and application of a tyrosine analogue, N-α-Fmoc-3-acetyl-l-tyrosine, for selective modification of proteins. As a model system, we synthesized the human immunodeficiency virus type 1 (HIV-1) Tat peptide (amino acids 47−56) containing the arginine rich RNA-binding region and replaced the Tyr-47 with 3-acetyl-tyrosine. The acetyl-Tyr-Tat peptide was subsequently labeled with a fluorescein derivative to study RNA−protein interactions by fluorescence energy transfer experiments. Our results showed that the Tat peptide binds to the rhodamine labeled TAR RNA with a dissociation constant (KD) of 1.0 ± 0.5 nM. This strategy of selective protein modification offers a versatile new procedure for labeling peptides of biological interest at a desired site when several nucleophilic side chains of lysine and cysteine are present. These methods would provide tools for postsynthetic peptide modification and introducing biophysical probes for structural and functional analysis of proteins

In Vivo Delivery of RNAi by Reducible Interfering Nanoparticles (iNOPs)

RNA interference (RNAi) has considerable potential as a therapeutic strategy, but the development of efficient in vivo RNA delivery methods remains challenging. To this end, we designed and synthesized chemically modified interfering nanoparticles (iNOPs) composed of functionalized poly-l-lysine dendrimers modified with reducible spacers to facilitate release of small interfering RNAs (siRNAs) in vivo. We show that the novel siRNA–iNOP complexes mediate efficient gene-specific RNAi in cultured cells and in mice, where they display enhanced tissue-targeting capabilities. At a clinically feasible dose of 1 mg kg^–1, apolipoprotein B (apoB) siRNA–iNOP complexes achieved ∼40–45% reduction of liver apoB mRNA and plasma apoB protein levels within 48 h of administration to mice, without apparent toxicity. Collectively, these findings demonstrate that siRNA delivery by the modified reducible iNOPs can provide a clinically significant and potentially tissue-specific new approach for RNAi therapy

Design and Creation of New Nanomaterials for Therapeutic RNAi

RNA interference is an evolutionarily conserved gene-silencing phenomenon that shows great promise for developing new therapies. However, the development of small interfering RNA (siRNA)-based therapies needs to overcome two barriers and be able to (i) identify chemically stable and effective siRNA sequences and (ii) efficiently silence target genes with siRNA doses that will be clinically feasible in humans. Here, we report the design and creation of interfering nanoparticles (iNOPs) as new systemic gene-silencing agents. iNOPs have two subunits: (i) a well-defined functionalized lipid nanoparticle as a delivery agent and (ii) a chemically modified siRNA for sustained silencing in vivo. When we injected iNOPs containing only 1−5 mg kg–1 siRNA into mice, an endogenous gene for apolipoprotein B (apoB) was silenced in liver, plasma levels of apoB decreased, and total plasma cholesterol was lowered. iNOP treatment was nontoxic and did not induce an immune response. Our results show that these iNOPs can silence disease-related endogenous genes in clinically acceptable and therapeutically affordable doses

Table S1 from Genome-Wide CRISPR Screen for Essential Cell Growth Mediators in Mutant KRAS Colorectal Cancers

Table with Gene level analysis of transduction replicates in the genome-wide xenograft screen using RIGER</p

Table S3 from Genome-Wide CRISPR Screen for Essential Cell Growth Mediators in Mutant KRAS Colorectal Cancers

Table showing the genes targeted in the secondary sgRNA library screen</p

Selective Binding of TAR RNA by a Tat-Derived β-Peptide

The interaction between the HIV-1 Tat protein and the TAR RNA element in the nascent viral genomic transcript is required for viral replication. An 11-residue β-peptide (1), an all-β homologue of the Arg-rich region Tat 47−57, binds TAR RNA with Kd = 29 ± 4 nM. A control β-peptide (2) in which all Arg side chains are replaced by Lys side chains shows increased affinity but decreased specificity for wild-type vs bulge-deleted TAR RNA, as do the α-peptide analogues of 1 and 2

Table S4 from Genome-Wide CRISPR Screen for Essential Cell Growth Mediators in Mutant KRAS Colorectal Cancers

Table with gene level analysis of transduction replicates in the secondary sgRNA library screen using STARS score</p

Supplementary Materials from Genome-Wide CRISPR Screen for Essential Cell Growth Mediators in Mutant KRAS Colorectal Cancers

This file contains supplemental figure legends, supplemental table titles, and supplemental materials and methods.</p