Inhibition of gene expression in human cells through small molecule-RNA interactions

Abstract

P rotein-nucleic acid interactions are involved in many cellular functions, including transcription, RNA splicing, and translation. Readily accessible synthetic molecules that can bind with high affinity to specific sequences of single-or double-stranded nucleic acids have the potential to interfere with these interactions in a controllable way, making them attractive tools for molecular biology and medicine. Successful approaches used thus far include duplex-forming (antisense) (1) and triplexforming (antigene) oligonucleotides (2-4), peptide nucleic acids (5), and pyrrole-imidazole polyamide oligomers (6, 7). Each class of compounds uses a readout system based on simple rules for recognizing the primary or secondary structure of a linear nucleic acid sequence. Another approach uses carbohydratebased ligands, calicheamicin oligosaccharides, which interfere with the sequence-specific binding of transcription factors to DNA and inhibit transcription in vivo (8, 9). Although antisense oligonucleotides and peptide nucleic acids use the familiar Watson-Crick base-pairing rules, two others, the triplex-forming oligonucleotides and the pyrrole-imidazole polyamides, take advantage of straightforward rules to read the major and minor grooves, respectively, of the double helix itself.

NMR-based identification of peptides that specifically recognize the d-arm of tRNA

“…There are many potential RNA targets, including RNA that is involved in cellular proteins interaction such as transcription, splicing, and translation and, RNA that is involved in viral infection such as human immunodeficiency virus (HIV). Several systematic screens have been performed against fragments of the HIV-1 genomic RNA, but in all cases, the high affinity hits were strongly cationic [1][2][3] and their recognition was dominated by electrostatic effects that are often poorly selective. There is thus a need for strategies based on high selectivity rather than on high affinity.…”

Discovery of a Small Molecule Tat-trans-Activation-responsive RNA Antagonist That Potently Inhibits Human Immunodeficiency Virus-1 Replication

“…Our approach entails covalent attachment of a dye molecule, disperse red, to the 5Ј end of TAR RNA (Scheme I) and incubation in a suspension of library beads prepared by the split-synthesis method (29). Although low molecular weight receptors can diffuse rapidly into a bead of TentaGel resin, we were not sure if a macromolecule such as a protein or large nucleic acid could diffuse to the bead interior where the bulk of the ligand molecule is displayed.…”

“…To evaluate the relative binding affinities of the parallel library compounds to TAR RNA, we measured their dissociation constants using a solid-phase assay as previously described (29). It is important to note that the K D values presented in this report should not be considered as absolute binding affinities that are usually obtained in homogeneous systems by biophysical methods.…”

“…The compounds were synthesized on the solid support as described earlier (36). After synthesis, the compounds were cleaved from the resin by trifluoroacetic acid treatment, purified by reverse phase HPLC, and the identity of the compounds was confirmed by matrix-assisted laser desorption ionization and fast atom bombardment mass spectrometry (29,36). The kinetics of HIV-1 NL4 -3 replication in Jurkat or MT4 cells was monitored by sampling culture supernatants every 3 or 4 days after infection and quantifying virus production by RT assay (41).…”

“…The suspension was centrifuged, and the equilibrium concentration of unbound RNA in the supernatant was determined by measuring its absorbance at 260 nm with a Shimadzu UV-1601 spectrometer. Knowing the initial concentrations of ligand and RNA, and assuming simple bimolecular receptor/substrate binding, the dissociation constant (K D ) was calculated from straightforward equations as described previously (29).…”