The peptidic compound, N-acetyl-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Cys(biotin)-NH2 (Tat10-biotin), contains the 9-amino acid sequence from the basic domain of the Tat protein responsible for specific interaction with TAR RNA. The cysteine residue provides an attachment site for biotin, which acts as a cellular uptake enhancer. Tat10-biotin binds a fragment of TAR RNA (deltaTAR) avidly and specifically, as measured in an electrophoretic gel shift assay. Tat10-biotin inhibited tat gene-induced expression of a stably transfected chloramphenicol acetyl transferase (CAT) reporter gene linked to the HIV-1 long terminal repeat (LTR) in a model cell assay, but did not inhibit phorbol ester-induced expression of CAT, thereby demonstrating a Tat-dependent mechanism of inhibition. Inhibition of HIV-1 replication after acute infection of MT2 cells was demonstrated by absence of HIV-induced syncytium formation and cytotoxicity, as well as by suppression of reverse transcriptase production. These results suggest that a peptide or peptide mimetic capable of competing with the TAR RNA-binding domain of Tat protein might be useful as a therapeutic agent for AIDS.

Two different methods were developed to prepare an adduct of a poly(ethylene glycol)-lysine copolymer with either cysteamine or 1-amino-2-methyl-2-propanethiol. Cysteine-containing peptides could then be disulfide-linked to the thiol groups on the polymer in a facile manner. In the described procedures, a coupling ratio of about 8 peptides/molecule of poly(ethylene glycol)-lysine copolymer (Mw = 27 000) was typically attained. The products were stable at neutral pH, but the peptides could be released from the polymer in a physiologically relevant reducing environment. The release rate was highly dependent on the linker used for forming the disulfide bond. To illustrate the potential biomedical usefulness of this polymer carrier, a Tat peptide-PEG conjugate was shown to inhibit expression of a reporter gene fused to the TAR element of human immunodeficiency virus in a model cell assay.

We observed a series of rapidly labeled U6 RNA bands, which were hybrid selected with U6 DNA, in nonirradiated human cells. The electrophoretic mobility of these bands in denaturing gels was lower than that of the known mature U6 RNA species, and was equivalent to transcripts up to approximately 7 nucleotides longer. These multiple U6 RNA species lost their label during a chase without a proportional increase in radioactivity in the known mature U6 RNA, which suggests that a substantial fraction is not processed into the major mature U6 RNA. During a label chase, the multiple U6 RNA bands appeared first in the cytoplasmic fraction and later in nuclei. One of the major rapidly labeled U6 RNA bands had the electrophoretic mobility of an RNA species one nucleotide shorter than the known mature U6 RNA. UV light induced a UV dose-dependent, preferential disappearance of recently synthesized molecules of the U6 RNA species of higher gel electrophoretic mobility, including the known mature U6 RNA. Since this effect was seen in cells pulse-labeled immediately before or after irradiation, it suggests that UV radiation induces the specific degradation of the electrophoretically faster moving species of U6 RNA, which are apparently shorter chains. The effect of UV light was RNA species-specific, was not seen in molecules synthesized long (e.g., 22 hr) before irradiation, and occurred in human and mouse cells.