A serum "sweet-doughnut" protein facilitates fibrosis evaluation and therapy assessment in patients with viral hepatitis.

Although liver fibrosis reflects disease severity in chronic hepatitis patients, there has been no simple and accurate system to evaluate the therapeutic effect based on fibrosis. We developed a glycan-based immunoassay, FastLec-Hepa, to fill this unmet need. FastLec-Hepa automatically detects unique fibrosis-related glyco-alteration in serum hyperglycosylated Mac-2 binding protein within 20 min. The serum FastLec-Hepa counts increased with advancing fibrosis and illustrated significant differences in medians between all fibrosis stages. FastLec-Hepa is sufficiently sensitive and quantitative to evaluate the effects of PEG-interferon-α/ribavirin therapy in a short post-therapeutic interval. The obtained fibrosis progression is equivalent to -0.30 stages/year in patients with sustained virological response, and 0.01 stages/year in relapse/nonresponders. Furthermore, long-term follow-up of the severely affected patients found hepatocellular carcinoma developed in patients after therapy whose FastLec-Hepa counts remained above a designated cutoff value. FastLec-Hepa is the only assay currently available for clinically beneficial therapy evaluation through quantitation of disease severity.

Characterization and application of a novel RNA aptamer against the mouse prion protein.

In order to isolate RNA aptamers against the mouse prion protein (mPrP), we carried out in vitro selection from RNA pools containing a 30-nucleotide randomized region. Aptamer 60-3 was found to have a high affinity for mPrP (K(d) = 5.6 +/- 1.5 nM), and 2'-fluoro-pyrimidine modifications for RNase resistance did not abolish its binding activity (K(d) = 22 +/- 4 nM). Following 5' biotinylation, aptamer 60-3 specifically detected PrP in mouse brain homogenate in a Northwestern blotting assay. To determine the mPrP-aptamer binding region, we performed protein-deletion-mutant analysis and competition-binding analysis using heparin. The results showed that aptamer 60-3 appears to have binding sites located between amino acids 23-108.

In vitro selection of RNA aptamers against cellular and abnormal isoform of mouse prion protein.

Prion disease is caused by conformational change of normal cellular type of prion protein (PrP(c)) folding into abnormal type (PrP(sc)). We succeeded to isolate anti-PrP(c) aptamers. In the presence of competitor RNA, anti-PrP(c) aptamers showed high affinity to PrP(c) (Kd = 10 nM). Heparin has prion binding affinity and partially interfered binding of the aptamer to PrP(c). 2'-Fluoro pyrimidine nucleotide modification still restored their binding affinity (Kd = 20 nM), which was applied for conventional dot- and western-blot assay like as antibody. We also succeeded to isolate anti-PrP(sc) aptamers appearing higher affinity to PrP(sc) in a dose-dependent manner. There is no sequence homology between those anti-PrP(c) and anti-PrP(sc) aptamers.

An RNA ligand inhibits hepatitis C virus NS3 protease and helicase activities.

The hepatitis C virus non-structural protein 3 (HCV NS3) possesses both protease and helicase activities that are essential for viral replication. In a previous study, we obtained RNA aptamers that specifically and efficiently inhibited NS3 protease activity (G9 aptamers). In order to add helicase-inhibition capability, we attached (U)14 to the 3'-terminal end of a minimized G9 aptamer, DeltaNEO-III. NEO-III-14U was shown to inhibit the NS3 protease activity more efficiently than the original aptamer and, furthermore, to efficiently inhibit the unwinding reaction by NS3 helicase. In addition, NEO-III-14U has the potential to diminish specific interactions between NS3 and the 3'-UTR of HCV-positive and -negative strands. NEO-III-14U showed effective inhibition against NS3 protease in living cells.

Designing and analysis of a potent bi-functional aptamers that inhibit protease and helicase activities of HCV NS3.

Nonstructural protein 3 (NS3) of hepatitis C virus (HCV) is a multi-functional enzyme having protease and helicase activities. NS3 is essential for HCV replication and proliferation. Previously, we obtained RNA aptamers against NS3 protease domain (Protease aptamer; deltaNEO-III and G9-II) and helicase domain (helicase aptamer; #5), and they inhibited the enzyme activities, respectively. We designed and constructed new bi-functional aptamers NEO-35-sX and G925-sX (X: 5-51 nts) by conjugating protease and helicase aptamers via oligo U spacer. Some of them showed 10-fold higher helicase inhibition than helicase aptamer #5 alone.

Structure/function analysis of an RNA aptamer for hepatitis C virus NS3 protease.

RNA aptamers that bind specifically to hepatitis C virus (HCV) NS3 protease domain (DeltaNS3) were identified in previous studies. These aptamers, G9-I, -II, and -III, were isolated using an in vitro selection method and they share a common loop with the sequence 5'-GA(A/U)UGGGAC-3'. The aptamers are potent inhibitors of the NS3 protease in vitro and may have potential as anti-HCV compounds. G9-I has a 3-way stem-loop structure and was selected for further characterization using site-directed mutagenesis. Mutations or deletions in stem-loop II do not interfere with binding or inhibition of DeltaNS3, but mutations or deletions in stem I and stem-loop III destroy the G9-I active conformation and interfere with inhibition of NS3 protease. A 51 nt fragment of 74 nt G9-I was identified (DeltaNEO-III) as is the minimal fragment of G9-I that is an effective inhibitor of the NS3 protease. Tertiary interactions involving functionally important nucleotides were identified in the active structure of G9-I using nucleotide analog interference mapping (NAIM). Strong interferences were focused in the conserved loop involving stem-loop III and stem I. For example, analog-interference caused at A(+8) and C(+24)-G(-36) base pair implied an A-minor motif involving the intramolecular base triple A(+8).C(+24)-G(-36), which is further supported by mutagenesis. These results suggested the interaction of stem I and stem-loop III is essential for the function of G9-I aptamer.

Inhibition of HCV NS3 protease by RNA aptamers in cells.

Non-structural protein 3 (NS3) of hepatitis C virus (HCV) has two distinct activities, protease and helicase, which are essential for HCV proliferation. In previous work, we obtained RNA aptamers (G9-I, II and III) which specifically bound the NS3 protease domain (DeltaNS3), efficiently inhibiting protease activity in vitro. To utilize these aptamers in vivo, we constructed a G9 aptamer expression system in cultured cells, using the cytomegarovirus enhancer + chicken beta-actin globin (CAG) promoter. By conjugating the cis-acting genomic human hepatitis delta virus (HDV) ribozyme and G9-II aptamer, a chimeric HDV ribozyme-G9-II aptamer (HA) was constructed, which was used to produce stable RNA in vivo and to create tandem repeats of the functional unit. To target the transcribed RNA aptamers to the cytoplasm, the minimal mutant of constitutive transport element (CTE), derived from type D retroviruses, was conjugated at the 3' end of HA (HAC). Transcript RNAs from (HA)(n) and (HAC)(n) were processed into the G9-II aptamer unit by the cis-acting HDV ribozyme, both in vitro and in vivo. Efficient protease inhibition activity of HDV ribozyme-G9-II aptamer expression plasmid was demonstrated in HeLa cells. Protease inhibition activity level of tandem chimeric aptamers, (HA)(n) and (HAC)(n), rose with the increase of n from 1 to 4.