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Biomedical subjects

P K Kumar

Publications and source records attributed to P K Kumar.

At least 19 recordsLinked to original sources
PubMed2000

Isolation and characterization of RNA aptamers specific for the hepatitis C virus nonstructural protein 3 protease.

Nonstructural protein 3 (NS3) from hepatitis C virus (HCV) is a serine protease that provides an essential function in maturation of the virus by cleaving the nonstructural regions of the viral polyprotein. The goal of this work was to isolate RNA aptamers that bind specifically to the NS3 protease active site in the truncated polypeptide DeltaNS3. RNA aptamers were selected in vitro by systematic evolution of ligands by exponential enrichment (SELEX). The RNA pool for SELEX had a 30-nucleotide randomized core region. After nine selection cycles, a pool of DeltaNS3-specific RNA aptamers were obtained. This RNA pool included 45 clones that divided into three main classes (G9-I, II and III). These classes include the conserved sequence GA(A/U)UGGGAC. These aptamers bind to DeltaNS3 with a binding constant of about 10 nM and inhibit approximately 90% of the protease activity of DeltaNS3 and MBP-NS3 (full-length of NS3 fused with maltose binding protein). In addition, these aptamers inhibited approximately 70% of the MBP-NS3 protease activity in the presence of the NS4A peptide P41. G9-I aptamer appeared to be a noncompetitive inhibitor for DeltaNS3 with a Ki approximately 100 nM in the presence of P41. These results suggest that the pool of selected aptamers have potential as anti-HCV compounds. Mutational analysis of the G9-I aptamer demonstrated that the sequences required for protease inhibition are in stem I, stem III and loop III of the aptamer. These regions include the conserved sequence GA(A/U)UGGGAC.

Base Sequence↗
PubMed1998

Selection of a RNA aptamer that binds to human activated protein C and inhibits its protease function.

A high-affinity RNA aptamer to human activated protein C (APC) was selected from a pool of random sequences using in vitro selection. Activated protein C, a trypsin-like serine protease plays an important role along with thrombin as a regulator in blood clotting cascade. After seven rounds of selection and amplification, a single predominant nucleic acid sequence APC-167, a 167-base oligonucleotide with a random sequence core of 120 bases, was obtained. The selected aptamer did not bind to thrombin or factor Xa and thus demonstrated specificity to APC. Furthermore, this aptamer was a non-competitive inhibitor to the cleavage reaction of a fluorogenic substrate catalyzed by APC. The inhibition constant (Ki) of APC-167 was 83 nM. The 99-base oligonucleotide (APC-99) derived from APC-167 by deleting both primer binding sites, was also found to inhibit APC strongly (Ki = 137 nM). Two stem-loop structures and at least one G x U wobble base pair in the stem were elucidated as important structural motifs for binding.

Base Composition↗
PubMed1997

Isolation of RNA aptamers specific to the NS3 protein of hepatitis C virus from a pool of completely random RNA.

Hepatitis C virus (HCV) is a single-stranded RNA virus and its genome is translated into a single large polyprotein. The viral-encoded NS3 protein possesses protease, nucleoside triphosphatase, and helicase activities. Since these activities appear to be important for viral replication, efforts are being made to identify compounds that might inhibit the enzymatic activities of NS3 and serve as potential anti-HCV agents. We used a genetic selection strategy in vitro to isolate, from a pool of completely random RNA (120 random bases), those RNA aptamers that could bind to NS3. After six cycles of selection and amplification, 14% of the pooled RNAs could bind specifically to the NS3 protein. When the aptamers in the pool (cycle 6) were analyzed for binding and inhibition of the proteolytic activity of NS3 with the NS5A/NS5B peptide as substrate (S1), two aptamers, designated G6-16 and G6-19 RNA, were found to inhibit NS3 in vitro. Kinetic studies of the inhibition revealed that the aptamer G6-16 inhibited the NS3 protease with an inhibitory constant (Ki) of 3 microM. We also analyzed aptamers G6-16 and G6-19 for their action with a longer protein substrate (amino acid region 2203-2506) and found that these aptamers efficiently inhibited the proteolytic activity of NS3. In addition, both G6-16 and G6-19 aptamers were found to inhibit the helicase activity of NS3. Since these aptamers possesses dual inhibitory function for NS3, they could prove to be useful as anti-HCV drug leads.

Base Sequence↗
PubMed1997

Inhibition of transcription by the TAR RNA of HIV-1 in a nuclear extract of HeLa cells.

Regulation of transcription of human immunodeficiency virus type-1 (HIV-1) requires specific interaction of Tat protein with the trans-activation response region (TAR). Inhibition of replication of HIV-1 has previously been achieved with a TAR decoy, namely a short RNA oligonucleotide that corresponded to the sequence of the authentic TAR RNA. Since TAR RNA has the potential to interact with cellular factors, we examined the effect of TAR RNA on efficiency of transcription in nuclear of HeLa cell extracts. We performed an in vitro transcription assay in the presence of authentic TAR RNA using a template that was driven by the CMV (cytomegalovirus) early promoter in a HeLa nuclear extract and found, for the first time, that TAR RNA inhibited transcription by approximately 60-70% independently of the Tat-TAR interaction. Furthermore, we evaluated inhibition of transcription by variants of TAR RNA and found that the TAR RNA loop, bases surrounding the loop, the triple base bulge and the 'lower' stem region of TAR RNA were responsible for the inhibition of transcription. Taken together, earlier reports on proteins that bind to TAR RNA and the present results suggest that integrity of TAR RNA is important for efficient binding to cellular transcription factors. As judged from the significant inhibition observed in this study, the TAR decoy might sequester transcription factors and thus it might potentially be able to inhibit transcription of housekeeping genes that are unrelated to Tat function.

Base Sequence↗
PubMed1997

Selection of RNA aptamers that bind specifically to the NS3 protease of hepatitis C virus.

The RNA genome of human hepatitis C virus (HCV) is translated into a large precursor polyprotein. The NS3 protease of HCV has a crucial role in the processing of the polyprotein into functional viral proteins. We have used an in vitro genetic-selection strategy to isolate high-affinity RNA aptamers that bind to the NS3 protein, especially to its protease domain. Starting from a RNA pool that had a random sequence core of 12-18 nucleotides, aptamers that bind specifically to the NS3 protein were selected after 10 rounds of selection and amplification. A single aptamer, 10G-1, was found predominantly (71%) in the selected pool. This aptamer could bind to the NS3 protein with a binding constant of 650 nM and inhibit the proteolytic activity in vitro. By phosphate-modification-interference analysis we showed that the phosphate residues that are critical for the binding of 10G-1 to NS3 lie within the selected regions of the aptamer and that binding involves electrostatic contacts with the phosphates of regions G28-U34 and A47-A55. The NS3-binding region in 10G-1 can serve as a basis for designing more potential inhibitors of the NS3 protein.

Base Sequence↗
PubMed1997

Expression of highly active recombinant NS3 protease domain of hepatitis C virus in E. coli.

The serine protease domain of HCV comprising amino acids 1027-1218 (deltaNS3) was expressed in E. coli with a His tag at its N-terminal end. The protease was purified to apparent homogeneity by a single step affinity chromatography resulting in high yields (approximately 3 mg/l of cultured cells). The deltaNS3 efficiently cleaves a 17-mer peptide corresponding to the NS5A-NS5B junction with kcat/Km = 160 x 10(-3) min(-1) microM(-1) in the presence of NS4A peptide. Our deltaNS3 represents the minimal domain possessing highly active protease of NS3 constructed so far. The deltaNS3 protein also efficiently processed a longer substrate corresponding to NS5A/5B junction (2203-2506 amino acids) that was synthesized by in vitro transcription and translation system.

Amino Acid Sequence↗
PubMed1997

Specific RNA aptamers to NS3 protease domain of hepatitis C virus.

In order to isolate RNA aptamers that bind specifically to NS3 protease domain (delta NS3) of hepatitis C virus, we carried out in vitro selection procedure using RNA pool that had 30 N random core region. After repeating nine cycles of selections and amplifications, a pool of RNAs that bind specifically to the delta NS3 were selected. A comparative analysis of 45 clones that were isolated from 9th cycle revealed three main classes that contain the conserved loop sequences GANUGGGAC. Moreover, the predominant class of aptamer (class I and III) appear to inhibit the protease activity efficiently.

Base Sequence↗
PubMed1996

Constructing an efficient trans-acting genomic HDV ribozyme.

We have engineered a genomic HDV ribozyme to construct several trans-acting ribozymes for use in trans to cleave target RNAs. Among the 10 different combinations attempted, only HDV88-Trans had cleavage activity on the 13-nucleotide substrate, R13, in vitro. To improve the cleavage efficiency, at least in vitro, of the HDV88-Trans ribozyme (kclv = 0.022 min(-1)), we have constructed several variants that differ in forming stem II (length) in the pseudoknot secondary structure model. When cleavage rate constants were analyzed and compared among variants of HDV88-Trans, HDV88-Trans-4 yielded kclv = 1.7 min(-1). HDV88-Trans-4 thus represents the highest active genomic HDV ribozyme that functions in trans thus far constructed, and has activity under physiological conditions (pH 7.1 at 37 degrees C with 1 mM of MgCl2).

Base Sequence↗
PubMed1996

Selection in vitro of trans-acting genomic human hepatitis delta virus (HDV) ribozymes.

In an effort to identify the functional structure as well as new active variants of the trans-acting genomic ribozyme of human hepatitis delta virus (HDV), we applied an in vitro selection procedure. A total of 14 rounds of selection and amplification was repeated and various mutant ribozymes in G10 and G14 pools analyzed. Active ribozymes which were isolated in the present study (from G10 and G14) all possessed conserved bases (that were identified earlier) in the cis-acting molecule. A dominant clone G10-68 variant was accumulated in generation 14. Interestingly, when base substitutions were analyzed in G10-68 variant, we found that this variant appears to be close to antigenome-like HDV ribozyme molecule. Further investigations of G10-68 confirmed that each mutated base was the most appropriate nucleotide at every position of the HDV ribozyme.

Base Sequence↗
PubMed1995

Suppression of testicular and epididymal functions in a non-human primate (bonnet monkey) by combined administration of a gonadotropin-releasing hormone antagonist and testosterone buciclate.

The ability of a long-acting androgen, testosterone buciclate (TB), to induce suppression of testicular and epididymal sperm functions when given in combination with a potent GnRH antagonist (Antide) either on day 1 or 45 of Antide administration (days 1-90) as well as the ability of TB to maintain Antide-induced suppression of spermatogenesis were evaluated in adult bonnet monkeys. A group of untreated animals (group I) acted as controls. All animals given Antide and androgen simultaneously (group II) became azoospermic but at different times. When androgen administration was delayed 45 days after start of Antide treatment (group III), the mean sperm concentration remained in the normospermic range and only three animals became azoospermic. Antide given alone (group IV) induced azoospermia in three animals and oligospermia in the remaining animals; spermatogenesis recovered when Antide was withdrawn and TB was injected. In all Antide-treated animals (groups II-IV), non-motile spermatozoa or sperm with non-progressive motility and poor gel penetrability were seen in the ejaculate.

Animals↗
PubMed1995

Effects of testosterone buciclate on testicular and epididymal sperm functions in bonnet monkeys (Macaca radiata)

The effects of testosterone buciclate (TB), a long-acting androgen ester given i.m. at four sites (20 mg/site) on days 1 and 91 of the study period (360 days), on reproductive and hormonal parameters were evaluated in five adult male bonnet monkeys; untreated animals (n = 5) acted as controls to monitor seasonal changes in these parameters. In control animals, testicular volume remained unchanged throughout the study; sperm count, motility and gel penetrability decreased while the percentage of spermatozoa showing retention of cytoplasmic droplet and coiled tail increased in June-July (days 210-240), preceded by reduction in serum testosterone (T) levels on days 120-150 (March-April). The TB-treated animals showed reduced testicular volume (days 90-270), suppressed sperm motility and gel penetrability (days 45-240 except on day 120), decreased sperm count (days 75-270), and an increased percentage of spermatozoa showing retention of cytoplasmic droplet and coiled tail (days 45-240 except on day 120). Even though serum T levels remained elevated until day 300, these levels were within the physiological range. The changes induced by TB were reversible. The suppression of testicular and epididymal functions by TB indicates that this long-acting androgen may have the potentiality to induce and maintain reversible sterility, but further evaluation needs to be carried out to develop an appropriate dosage regimen that would prevent return to normal functions in order to develop this long-acting androgen as a hormonal male contraceptive.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Artificial evolution and natural ribozymes.

In vitro selection techniques have been used to probe the sequence, structure, and function of natural ribozymes such as the viroid hammerheads and group I self-splicing introns. These artificial evolution experiments help to delimit the range of alternative structures and functions that are available to catalytic RNAs, and thus can provide insights into why particular sequences or mechanisms were fixed during the course of natural selection. Further, the wide variety of forms and functions that ribozymes have been found to assume in the laboratory provides inferential support for the hypothesis that much of modern metabolism may have been distantly derived from biochemistry centered on RNA rather than protein catalysts.

Animals↗
PubMed1995

In vitro selection analysis of trans-acting HDV ribozyme.

In order to identify the functional structure as well as new active variants of the trans-acting genomic ribozyme of human hepatitis delta virus (HDV), we applied an in vitro selection procedure. After 10 generations, a randomized pool of trans-acting ribozymes accumulated in which the secondary structure of each ribozyme confirmed to the pseudoknot model and important bases in single-stranded regions were all conserved. We were surprised that mutated ribozymes derived from genomic sequence were changed to anti-genomic-like sequences. Further investigations of the most active variant confirmed that each mutated base was the most appropriate nucleotide at every position of HDV ribozyme.

Base Sequence↗
PubMed1995

In vitro selection of RNA aptamers that can bind specifically to Tat protein of HIV-1.

The Tat protein of human immunodeficiency virus interacts specifically with its target RNA sequence, TAR, and activates viral gene expression at the early stage of infection. Here, we have used in vitro genetic selection strategy to determine what sequence or structural motifs might exist between RNA's that interact specifically with the Tat protein. Starting from a RNA pool that has a 120 base random sequence core, aptamers that bind specifically to the Tat protein were selected by repeating 11 rounds of selection and amplification. A comparative analysis of 64 aptamers that were isolated from the 11th generation revealed two main sequence classes. Interestingly, one of these two classes of aptamers had minimum of one U residues in bulge loop and 2 specific adjacent base pairs. This region is very much homologous to the core sequence of TAR RNA that is essential for the specific Tat-TAR interactions. Further analyses of the sequences from the 11th generation should reveal what kind of RNA structures are required in order to show a high affinity for the Tat protein.

Base Sequence↗
PubMed1994

Identification of phosphate oxygens that are important for self-cleavage activity of the HDV ribozyme by phosphorothioate substitution interference analysis.

A phosphorothioate substitution interference assay was used to investigate the role of the pro-Rp oxygens of phosphate groups in the self-cleavage reaction of the genomic human hepatitis delta virus (HDV) ribozyme. Incorporation of several different phosphorothioates (NTP alpha S) into the HDV ribozyme inhibited the self-cleavage activity. Incorporation of uridine 5' phosphorothioate or adenosine 5' phosphorothioate maintained 72% of the original self-cleavage activity whereas incorporation of guanosine 5' phosphorothioate or cytosine 5' phosphorothioate into the precursor reduced self-cleavage activity to about 20% in each case. Using partially substituted phosphorothioate-modified transcripts, we identified the pro-Rp oxygens that are important for the ribozyme activity, and they are located at positions 0, 1, 4, 5, 21, 24, 25, 27, 28, 30-34, 40, 43 and 75. In particular, the pro-Rp oxygens at positions 0, 1 and 21 are appear to be critical for the self-cleavage activity of the HDV ribozyme.

Base Sequence↗
PubMed1994

Chemical probing studies of variants of the genomic hepatitis delta virus ribozyme by primer extension analysis.

We have investigated in detail the higher order structure of the genomic hepatitis delta virus (HDV) ribozyme using various base-specific chemical probes under native, semi-denaturing, and denaturing conditions. The bases of the HDV ribozyme were probed by treatment with dimethyl sulfate [which reacts with A (at N1) and C (at N3)] and a carbodiimide [which reacts with U (at N3) and G (at N1)]. In addition, for probing G residues (at N7), RNA samples were treated with NaBH4 and aniline after modification by treatment with dimethyl sulfate. The sites of modified positions were identified by primer extension analysis with reverse transcriptase. In general, our results are consistent with the proposed pseudoknot model of secondary structure, a model that is based on data from ribonucleolytic cleavage experiments. Our results provide clues to the identification of interacting bases in the HDV ribozyme. Furthermore, using this method we identified local conformational changes in several stem variants.

Aniline Compounds↗
PubMed1993

Enhancement of the cleavage rates of DNA-armed hammerhead ribozymes by various divalent metal ions.

In order to characterize structure-function relationships, the kinetic behavior of chimeric RNA/DNA ribozyme was compared with that of all RNA ribozyme. Determined kcat values were proven to represent the chemical-cleavage step and not the product-dissociation step. In agreement with the finding by Dahm and Uhlenbeck [Biochemistry 30, 9464-9469 (1991)], various metal ions, including Co2+ and Ca2+ with the ionic radius of 0.65 and 1.0 A, respectively, could support hammerhead cleavage for both types of ribozyme. Measurements of kinetic parameters in the presence of various divalent metal ions revealed that DNA arms always enhanced kcat values. Chemical-probing data using dimethylsulfate indicated that the catalytic-loop structures of all-RNA and chimeric ribozymes were nearly identical with the exception of enhanced termination of primer extension reactions at C3 in the case of the chimeric ribozyme. These observations and others demonstrate that DNA substitution in non-catalytic-loop regions increases chemical-cleavage activity, possibly with an accompanying very subtle change in the structure.

Base Sequence↗