SSCP screening of individual aptamers.

Aptamers are specific binding nucleic acids that emerge from in vitro selection. During the systematic evolution of ligands by exponential enrichment (SELEX) procedure, analysis of the sequences of the numerous selected individual molecules becomes an important step in the final stage of aptamer selection. The sequencing of cloned aptamers from the selected pool generally reveals groups of identical sequences and rarely occurring individual aptamers. This study demonstrates an approach similar to the single strand conformation polymorphism (SSCP) method used for mutation testing in genes. Human angiotensin I-specific aptamers have been used to show the efficiency of the SSCP method to classify selected individual sequences into identity groups, which minimizes sequencing efforts. Additionally this approach allows the rapid isolation and identification of aptamers from a mixture.

Long-term expression of foreign genes in normal human epidermal keratinocytes after transfection with lipid/DNA complexes.

Normal human epidermal keratinocytes were isolated and cultivated in serum-free medium. The expression of the integrin subunits alpha6 and beta1 indicated that a high number of keratinocytes from the stem cell system was present. These cells were transfected with complexes made of different cationic lipids and marker genes. Effectene showed a 20-fold higher transfection efficiency, compared to Lipofectin and Lipofectamine, and a similar low toxicity. The transfection protocol was optimised. A DNA/lipid ratio of 0.133 showed the highest transfection efficiency. Keratinocytes expressed the marker gene luciferase for 20 days. The maximum expression occurred after 3-4 days, where individual patches of fluorescent keratinocytes were detected. Transfected keratinocytes, cultivated at the air-liquid interface, expressed the marker gene beta-galactosidase for at least 7 weeks.

Endogenous fragment of hemoglobin, neokyotorphin, as cell growth factor.

It is shown that neokyotorphin (the alpha-globin fragment 137-141) stimulates proliferation of normal cells (murine embryonic fibroblasts, red bone marrow and spleen cells) and tumor cells (murine melanoma and transformed fibroblasts L929) in the absence or in the presence of fetal bovine serum. In contrast to serum deprivation conditions, the ability to potentiate L929 cell growth in the presence of fetal serum is strongly cell density dependent. The peptide also enhances the viability of L929 cells, murine embryonic fibroblasts and of the primary cultures of murine red bone marrow cells and splenocytes under serum-deprivation conditions for at least 72 h. The results of flow cytometry analysis suggest that the effect of neokyotorphin on survival of L929 cells in serum-free culture medium is due to maintenance of cell proliferation in the absence of growth factors. Along with cell cycle progression the peptide induces reversible reduction of L929 cell size.

Transfection of normal human epidermal keratinocytes with lipid/dna complexes in vitro.

Highly proliferative normal human epidermal keratinocytes (NHK) were isolated from human foreskin biopsies, cultivated in serum-free medium and characterized by flow cytometry. The expression of cytokeratin 19, cytokeratin 14 and vimentin indicated that the suspension contained a high percentage of undifferentiated cells of the basal epidermal layer. The NHK were transfected in vitro with lipid/DNA complexes made of Effectene or Lipofectamine and different reporter genes. The transfection efficiency of Effectene/DNA complexes was 20fold higher compared to Lipofectamine. Transfected keratinocytes continued to grow and developed within 2 weeks a cellular multilayer (3-D culture). Areas of transfected cells were detected within this layer.

Basic properties of populations generated in the frame of one-parameter discrete model of genetic diversity.

Previously, when discussing the properties of one parameter discrete model of genetic diversity (M.Yu. Shchelkanov et al, J. Biomol. Struct. Dyn. 15, 887-894 (1998)), we took into account Hamming distance distribution only between precursor and arbitrary descendant sequences. However, really there are sets of sequence populations produced during amplification process. In the presented work we have investigated Hamming distance distributions between sequences from different descendant sets produced in the frame of one parameter discrete model. Two basic descendant generation operators (so called amplifiers) are introduced: 1) the last generation amplifier, L, which produces descendants with precursor elimination; 2) all generations amplifier, G, which produces descendants without precursor elimination. Generalization of one-parameter discrete model for the case when precursor sequences do not coincide are carried out. Using this generalization we investigate the distribution of Hamming distances between L- and G-generated sequences. Basic properties of L and G operators, L/G-choice alternative problem have been discussed. Obtained results have common theoretical significance, but they are more suitable for high level genetic diversity process (for example, HIV diversity).

A peptide construct containing B-cell and T-cell epitopes from the foot-and-mouth disease viral VP1 protein induces efficient antiviral protection.

A new peptide construct Palm135-158-GGA-170-188(Acm) has been synthesized and investigated in a number of in vitro and in vivo test systems. The construct contains a virus specific T-helper epitope within the 170-188 sequence of VP1, in addition to the main antigenic 135-158 region of the foot-and-mouth disease viral VP1 protein (strain A22). The construct has higher protective, antigenic, immunogenic and T-cell proliferative activity then the previously described shorter peptide Palm(2)135-159. The 170-188 part of the construct serves as a virus specific T-epitope, responsible for the enhanced immunogenic and protective activity of the construct.

Integrin receptor-targeted transfer peptides for efficient delivery of antisense oligodeoxynucleotides.

Integrin receptor-targeted transfer of oligodeoxynucleotides (ODNs) by small synthetic peptides was used for improving delivery of antisense ODNs. An 18-mer phosphodiester bond containing ODN complementary to c-myb-encoded mRNA was complexed with several transfer peptides, containing as their parts two modules: (a) an RGD-motif as targeting sequence for integrin receptor and (b) nucleocapsid protein (NCp) 7 of HIV-1 or NCp7-derived peptides for complex formation with the ODNs. The amount of antisense ODN required for the inhibition of proliferation of human myeloid cell line HL-60 in vitro can be more than 50-fold reduced by complexing with transfer peptides. The efficiency of antisense delivery was increased by multimerization of the targeting sequence for the integrin receptor. Competition with integrin peptide abolished the effect, indicating that the integrin receptor is indeed responsible for the reaction.

Amino acid requirements of the nucleocapsid protein of HIV-1 for increasing catalytic activity of a Ki-ras ribozyme in vitro.

The nucleocapsid protein NCp7 of HIV-1 is a single-stranded nucleic acid binding protein with several functions such as specific recognition, dimerization and packaging of viral RNA, tRNA annealing to viral RNA and protection against nucleases. Since some of these functions involve annealing and double-stranded RNA-melting activity we applied the nucleocapsid protein to a hammerhead ribozyme specific for the activated Ki-ras mRNA in vitro, which carries at its mutated codon 12 a GUU site. A synthetic ribozyme containing 2'-O-allyl-modified nucleotides and alternatively in vitro transcribed ribozymes were used. At a one to one molar ratio of substrate to ribozyme almost no cleavage is observed at 37 degrees C. Presence of a synthetic nucleocapsid protein significantly increases the catalytic activity of the ribozyme. Kinetic analyses by means of single and multiple turnover reactions performed at various substrate to ribozyme ratios lead to only a slight stimulation of the rate constants for single turnover reactions. The rate constants in multiple turnover reactions, however, are stimulated up to 17-fold by the presence of the nucleocapsid protein. The activating region of the nucleocapsid protein was characterized by a number of mutants. The mutants demonstrate that activation requires both basic amino acid clusters as evidenced by point mutations. Deletion mutants indicate that the second zinc finger is totally dispensable and that replacement of the first zinc finger by a glycine-glycine spacer only slightly reduces the enhancing effect of the nucleocapsid protein on the ribozyme.

Specific binding of HIV-1 nucleocapsid protein to PSI RNA in vitro requires N-terminal zinc finger and flanking basic amino acid residues.

The nucleocapsid (NC) protein of human immunodeficiency virus HIV-1 (NCp7) is responsible for packaging the viral RNA by recognizing a packaging site (PSI) on the viral RNA genome. NCp7 is a molecule of 55 amino acids containing two zinc fingers, with only the first one being highly conserved among retroviruses. The first zinc finger is flanked by two basic amino acid clusters. Here we demonstrate that chemically synthesized NCp7 specifically binds to viral RNA containing the PSI using competitive filter binding assays. Deletion of the PSI from the RNA abrogates this effect. The 35 N-terminal amino acids of NCp7, comprising the first zinc finger, are sufficient for specific RNA binding. Chemically synthesized mutants of the first zinc finger demonstrate that the amino acid residues C-C-C/H-C/H are required for specific RNA binding and zinc coordination. Amino acid residues F16 and T24, but not K20, E21 and G22, located within this zinc finger, are essential for specific RNA binding as well. The second zinc finger cannot replace the first one. Furthermore, mutations in the basic amino acid residues flanking the first zinc finger demonstrate that R3, 7, 10, 29 and 32 but not K11, 14, 33 and 34 are also essential for specific binding. Specific binding to viral RNA is also observed with recombinant NCp15 and Pr55Gag. The results demonstrate for the first time specific interaction of a retroviral NC protein with its PSI RNA in vitro.

Conformational and nucleic acid binding studies on the synthetic nucleocapsid protein of HIV-1.

A 55 residue peptide corresponding to the nucleocapsid protein of HIV-1 (NCp7) containing two zinc binding domains as well as three truncated peptides were synthesized by Fmoc-based solid phase synthesis using the fragment condensation approach. Circular dichroism (CD) data support a conformational model in trifluoroethanol/buffer solution consisting of two helical segments at the chain ends with two Zn-modules in the center of the molecule. CD titration experiments show that the synthetic protein binds two equivalents of Zn2+ stoichiometrically, and the Zn2+ induced conformational changes are completely reversible by addition of EDTA. NCp7 and its S-acetamidomethylated analog (NCp7-Acm), devoid of the zinc co-ordination centers, exhibit preferential binding to RNA with a Kd = approximately 10(-9) M irrespective of the cysteine modification as determined by filter binding assays. The binding affinity of the NCp7 protein to single-stranded DNA is lower than to RNA. Binding to double-stranded DNA is lower than to ssDNA. The NCp7-Acm protein exhibits reduced single-stranded DNA binding affinity compared to the unmodified protein. Nucleic acid binding analyses with the fragments of NCp7 protein suggest that two basic amino acid stretches are involved in RNA binding of the NCp7.

Nucleocapsid protein of HIV-1 and its Zn2+ complex formation analysis with electrospray mass spectrometry.

The Zn2+ binding properties of the synthetic nucleocapsid protein (Ncp7) of HIV-1, containing two zinc-binding domains, have been studied using electrospray mass spectrometry (ES-MS). ES-MS measurements revealed strong binding of Zn2+ by Ncp7. Its shorter fragments, Ncp7-(1-35)- and (29-55)-peptides, each containing only one zinc-binding domain, bind one equivalent of Zn2+ ions tightly. ES-MS studies allows these fragments to be distinguished in terms of their binding affinity: they showed stronger binding of Zn2+ by Ncp7-(1-35)-peptide. Surprisingly, in addition to the expected two zinc-binding domains, a third metal binding site was detected in Ncp7. However, this site appears to bind different metal ions without selectivity and most probably reflects salt formation at the C-terminal acidic residues.