Isolation and Characterization of 2'-amino-modified RNA Aptamers for Human TNFα

Human tumor necrosis factor a (hTNFa), a pleiotropic cytokine with activities ranging from host defense mechanisms in infection and injury to severe toxicity in septic shock or other related diseases, is a promising target for drug screening. Using the SELEX (systematic evolution of ligands by exponential enrichment) process, we isolated oligonucleotide ligands (aptamers) with high affinities for hTNFa. Aptamers were selected from a starting pool of 40 randomized sequences composed of about 1015 RNA molecules. Representative aptamers were truncated to the minimal length with high affinity for hTNFa and were further modified by replacement of 2'-OH with 2'-F and 2'-NH2 at all ribopurine positions. These modified RNA aptamers were resistant to nuclease. The specificity of these aptamers for hTNFa was confirmed, and their activity to inhibit the cytotoxicity of hTNFa on mouse L929 cells was determined. Results demonstrated that four 2'-NH2-modified aptamers bound to hTNFa with high affinity and blocked the     

人源性抗TNFα小分子抗体的改构和分析

在获得人源性抗人TNF α单链抗体 (ScFv)基因序列的基础上 ,对ScFv的连接肽部分进行基因改造 ,并构建了Fab抗体基因。改构前后的ScFv分别重组入表达载体pBV2 2 0 ,经 42℃热诱导 ,在E .coliDH5α中表达了ScFv蛋白 ,得到分子量约为 30kD的重组蛋白质 ,改构前后ScFv的表达量分别占菌体总蛋白质的 6 .5 %和13 .8%。同时构建Fab可溶性表达载体并转化非抑制型菌株HB2 15 1,经IPTG诱导 ,在约 5 0kD分子量处呈现一条新生蛋白质条带。从大肠杆菌裂解液中对ScFv进行了复性和层析纯化 ,对Fab基因的表达产物进行了亲和层析纯化 ,并证实 :(1)改构后ScFv在大肠杆菌中的表达量有所提高 ;(2 )改构前后的ScFv与Fab均具有与hTNF α相结合的活性 ,具有GGGGS连接肽的ScFv与hTNF α的亲和常数为 6 .70× 10 4 (mol/L) -1,而改构后具有(GGGGS) 3 连接肽的ScFv的亲和常数提高为 7.2 7× 10 5(mol/L) -1,Fab与hTNF α的亲和常数为 7.6 1× 10 5(mol/L) -1,Fab与改构后ScFv的亲和力无明显差异 ;(3)ScFv与Fab均有中和hTNF α细胞毒的作用 ,具有(GGGGS) 3 连接肽的ScFv与Fab的中和活性基本相同 ,但均明显低于一株鼠源性单抗     

Isolation and characterization of enantioselective DNA aptamers for ibuprofen

Single stranded DNA aptamers that can bind to ibuprofen, a widely used anti-inflammation drug, were selected from random DNA library of 10¹⁵ nucleotides by FluMag-SELEX process. Five different sequences were selected and their enantioselectivity and affinity were characterized. Three out of five aptamer candidates did not show any affinity to (S)-ibuprofen, but only to racemic form of ibuprofen, suggesting that they are (R)-ibuprofen specific aptamers. Another two aptamer candidates showed affinity to both racemic form and (S)-ibuprofen, which were considered as (S)-ibuprofen specific aptamers. The affinity of five ssDNA aptamers isolated was in a range of 1.5–5.2 μM. In addition, all of these five aptamers did not show any affinity to analogues of ibuprofen in its profen’s group (fenoprofen, flubiprofen, and naproxen) and the antibiotics of oxytetracycline, another control.     

The generation and characterization of antagonist RNA aptamers to human oncostatin M

Oncostatin M (OSM) is a multifunctional member of the interleukin-6 cytokine family. OSM has been implicated as a powerful proinflammatory mediator and may represent a potentially important, novel therapeutic opportunity for treatment of established rheumatoid arthritis. To further investigate the role of OSM in inflammatory disorders, we have isolated a series of RNA aptamers that bind specifically to human OSM. The highest affinity aptamer, designated ADR58, has been characterized in a series of in vitro and cell based assays. ADR58 has an affinity of 7 nm for human OSM, and it can antagonize OSM binding to the gp130 receptor and specifically antagonize OSM mediated signaling. The aptamer has been truncated in length to 33 bases, all pyrimidine positions are substituted with 2' fluorine, and 14 of 18 purine positions have been substituted with 2' O-methyl to increase stability toward nucleases. This truncated, modified form of ADR58 retains complete affinity and functional activity for OSM. This aptamer may be used as a tool to further investigate the role of OSM in inflammatory disorders and may also have role as a therapeutic agent.     

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.     

Production and characterization of RNA aptamers specific for amyloid fibril epitopes

One of the most fascinating features of amyloid fibrils is their generic cross-beta architecture that can be formed from many different and completely unrelated proteins. Nonetheless, amyloid fibrils with diverse structural and phenotypic properties can form, both in vivo and in vitro, from the same protein sequence. Here, we have exploited the power of RNA selection techniques to isolate small, structured, single-stranded RNA molecules known as aptamers that were targeted specifically to amyloid-like fibrils formed in vitro from beta(2)-microglobulin (beta(2)m), the amyloid fibril protein associated with dialysis-related amyloidosis. The aptamers bind with high affinity (apparent K(D) approximately nm) to beta(2)m fibrils with diverse morphologies generated under different conditions in vitro, as well as to amyloid fibrils isolated from tissues of dialysis-related amyloidosis patients, demonstrating that they can detect conserved epitopes between different fibrillar species of beta(2)m. Interestingly, the aptamers also recognize some other, but not all, amyloid fibrils generated in vitro or isolated from ex vivo sources. Based on these observations, we have shown that although amyloid fibrils share many common structural properties, they also have features that are unique to individual fibril types.     

High-affinity and specific recognition of human thyroid stimulating hormone (hTSH) by in vitro-selected 2'-amino-modified RNA.

RNA sequences containing 2'-amino pyrimidines that bind with high-affinity to human thyroid stimulating hormone (hTSH) were isolated from a random sequence library by an in vitro selection-amplification procedure. A representative RNA ligand (T-15) has an equilibrium dissociation constant (Kd) of 2.5 nM for its interaction with hTSH and can discriminate between other members of the glycohormone family; no detectable binding was observed at low micromolar concentrations of hCG (human chorionic gonadotropin), while measured Kd values for the interactions with hLH (human leutinizing hormone) and hFSH (human follicle stimulating hormone) were > 1 microM and approximately 0.2 microM, respectively. The detection of hTSH in a dot blot assay with radiolabeled T-15 RNA was demonstrated.     

Isolation and characterization of high affinity aptamers against DNA polymerase iota

Human DNA-polymerase iota (Pol ι) is an extremely error-prone enzyme and the fidelity depends on the sequence context of the template. Using the in vitro systematic evolution of ligands by exponential enrichment (SELEX) procedure, we obtained an oligoribonucleotide with a high affinity to human Pol ι, named aptamer IKL5. We determined its dissociation constant with homogenous preparation of Pol ι and predicted its putative secondary structure. The aptamer IKL5 specifically inhibits DNA-polymerase activity of the purified enzyme Pol ι, but did not inhibit the DNA-polymerase activities of human DNA polymerases beta and kappa. IKL5 suppressed the error-prone DNA-polymerase activity of Pol ι also in cellular extracts of the tumor cell line SKOV-3. The aptamer IKL5 is useful for studies of the biological role of Pol ι and as a potential drug to suppress the increase of the activity of this enzyme in malignant cells.