Penmetcha K. R. Kumar

Molecular beacon aptamer fluoresces in the presence of Tat protein of HIV‐1

We reported an aptamer, RNATat that binds to the Tat protein of HIV with two orders of magnitude greater (133‐fold) affinity over the TAR RNA of HIV‐1 and specifically inhibits the Tat‐dependent trans‐activation of transcription, both in vitro and in vivo (demonstrated in the accompanying article, Yamamoto et al., this issue pp. 371–388). We now report the use of aptamer‐derived oligomers to analyze the Tat of HIV and the possible applications of such constructs in the field of biosensors.

A novel RNA motif that binds efficiently and specifically to the Tat protein of HIV and inhibits the trans‐activation by Tat of transcription in vitro and in vivo

To find a novel RNA that would bind efficiently and specifically to Tat protein but not to other cellular factors, we used an in vitro selection method and isolated a novel aptamer RNATat, a 37‐mer RNA oligomer, that binds efficiently to the Tat protein of HIV‐1. In the present study, we analysed various properties of aptamer RNATat, including binding kinetics, identification of functional groups for Tat binding, and inhibition of Tat function.

Aptamer That Binds to the gD Protein of Herpes Simplex Virus 1 and Efficiently Inhibits Viral Entry

ABSTRACT The ectodomain of the gD protein of herpes simplex viruses (HSVs) plays an important role in viral entry by binding to specific cellular coreceptors and mediating viral entry to the host cells. In the present study, we isolated RNA aptamers (aptamer-1 and aptamer-5) that specifically bind to the gD protein of HSV-1 with high affinity and are able to discriminate the gD protein of a different virus, HSV-2. Aptamer-1 efficiently interfered with the interaction between the gD protein and the HSV-1 target cell receptor (HVEM) in a dose-dependent manner. The 50% effective concentration (EC50) of aptamer-1 was estimated to be in the nanomolar range (60 nM). Furthermore, aptamer-1 was analyzed for anti-HSV-1 activity by using plaque assays, and it efficiently inhibited viral entry with an estimated Ki of 0.8 μM. To expand the future applications of aptamer-1, a shorter variant was designed by using both mapping and boundary analyses, resulting in the mini-1 aptamer (44-mer). Compared to the full-length aptamer, mini-1 had at least as high an affinity, specificity, and ability to interfere with gD-HVEM interactions. These studies suggest that the mini-1 aptamer could be explored further as an anti-HSV-1 topical therapy designed to prevent the risk of acquiring HSV-1 infection through physical contact.

Monitoring biomolecular interactions on a digital versatile disk: a BioDVD platform technology.

A spinning-disk biosensor utilizing optical interference of reflected light from a multilayered structure, consisting of dielectric, metal, and optical phase-change thin films, is shown to have the potential to monitor various interactions on its surface. We refer to this platform as a BioDVD, since it utilizes the optical system of a digital versatile disk (DVD) to measure changes in reflected light intensity. Here, we demonstrated that nucleic acid hybridization and RNA-protein interactions can be analyzed efficiently, in a label-free environment, by measuring the reflected light intensity using a DVD-like mechanism. Moreover, our studies revealed that the detection sensitivity for the interactions on the BioDVD can be altered by shifting the state of the phase-change materials, where the amorphous state can be used for analysis and another state (crystalline) can be used both for recording information and selectively masking areas of the disk.

Human vault-associated non-coding RNAs bind to mitoxantrone, a chemotherapeutic compound

Human vaults are the largest cytoplasmic ribonucleoprotein and are overexpressed in cancer cells. Vaults reportedly function in the extrusion of xenobiotics from the nuclei of resistant cells, but the interactions of xenobiotics with the vault-associated proteins or non-coding RNAs have never been directly observed. In the present study, we show that vault RNAs (vRNAs), specifically the hvg-1 and hvg-2 RNAs, bind to a chemotherapeutic compound, mitoxantrone. Using an in-line probing assay (spontaneous transesterification of RNA linkages), we have identified the mitoxantrone binding region within the vRNAs. In addition, we analyzed the interactions between vRNAs and mitoxantrone in the cellular milieu, using an in vitro translation inhibition assay. Taken together, our results clearly suggest that vRNAs have the ability to bind certain chemotherapeutic compounds and these interactions may play an important role in vault function, by participating in the export of toxic compounds.

A sensitive multilayered structure suitable for biosensing on the BioDVD platform.

Several technologies are currently available for the analysis of biomolecular interactions with high sensitivity and efficiency. However, these instruments are invariably expensive and, thus, are not suitable for bedside analyses. To circumvent this issue, we have previously reported a BioDVD platform that allowed us to use a DVD mechanism to monitor various biomolecular interactions [Gopinath et al., 2008, ACS Nano 2, 1885-1895]. In the present study, to improve the sensitivity of the BioDVD platform for various analyses, we have performed computer simulations to optimize the ZnS-SiO(2) layer thicknesses and determined an optimized optical interferometric response after adjusting the ZnS-SiO(2) layer thickness to 65 and 60 nm for the inner and outer layer thicknesses, respectively. Biomolecular interaction analyses performed with the optimized BioDVD disks revealed a 3-fold improvement in the sensitivity, compared to our previously reported multilayered structure. In this study, we have also shown that the BioDVD platform is suitable not only for analyzing nucleic acid hybridization and interactions between RNA-small ligands and RNA-proteins, but also for antigen-antibody interactions. Furthermore, our evaluations revealed that each sample required no more than 10 tracks of data to analyze the biomolecular interactions on the BioDVD platform, which permits a greater number of spots per BioDVD disk and also reduces the time needed to measure the biomolecular interactions.

Influence of nanometric holes on the sensitivity of a waveguide-mode sensor: Label-free nanosensor for the analysis of RNA aptamer-ligand interactions

Evanescent-field-coupled (EFC) waveguide-mode sensors can be used to detect nucleic acids or proteins from the changes in the local index of refraction upon adsorption of the target molecule on a waveguide surface. We recently described an EFC waveguide-mode sensor in which nanometric holes on a waveguide film resulted in an improved sensitivity in the analysis of the interactions of biomolecules. In the present study, we have shown that sensitivity depends upon the diameter of the holes, where increase in diameter of holes increases spectral shift resulting in an improved sensitivity. Using this improved EFC waveguide-mode sensor, we could detect interactions between RNA and a small ligand, cyanocobalamin (vitamin B 12), and between RNA and a protein (human coagulation factor IXa). These two interactions were monitored on surfaces modified with biotin-streptavidin-biotin and N-(2-trifluoroethanesulfonatoethyl)- N-(methyl)triethoxysilylpropyl-3-amine, respectively.

A potent anti-coagulant RNA aptamer inhibits blood coagulation by specifically blocking the extrinsic clotting pathway

A potent antidote-controlled aptamer, as an anticoagulant, has reportedly overcome the complications of acute bleeding by the administration of available anticoagulants. In the present study, we provide evidence that the aptamer binds specifically to factors IX and IXa and inhibits their functions. Furthermore, we demonstrated that the aptamer inhibits blood coagulation by interfering with the extrinsic pathway, blocking the complex of factor VIIa and tissue factor interactions with factor IX. The results from the previous and present studies suggest that the aptamer probably binds in the vicinity of the EGF1 and EGF2 domains of factor IX.

Structural basis of HutP-mediated anti-termination and roles of the Mg2+ ion and L-histidine ligand.

HutP regulates the expression of the hut structural genes of Bacillus subtilis by an anti-termination mechanism and requires two components, Mg2+ ions and l-histidine. HutP recognizes three UAG triplet units, separated by four non-conserved nucleotides on the terminator region. Here we report the 1.60-Å resolution crystal structure of the quaternary complex (HutP–l-histidine–Mg2+–21-base single-stranded RNA). In the complex, the RNA adopts a novel triangular fold on the hexameric surface of HutP, without any base-pairing, and binds to the protein mostly by specific protein–base interactions. The structure explains how the HutP and RNA interactions are regulated critically by the l-histidine and Mg2+ ion through the structural rearrangement. To gain insights into these structural rearrangements, we solved two additional crystal structures (uncomplexed HutP and HutP–l-histidine–Mg2+) that revealed the intermediate structures of HutP (before forming an active structure) and the importance of the Mg2+ ion interactions in the complexes.

Snake-venom-derived Factor IX-binding protein specifically blocks the γ-carboxyglutamic acid-rich-domain-mediated membrane binding of human Factors IX and X

A potent anticoagulant protein, IX-bp (Factor IX binding protein), has been isolated from the venom of Trimeresurus flavoviridis (habu snake) and is known to bind specifically to the Gla (gamma-carboxyglutamic acid-rich) domain of Factor IX. To evaluate the molecular basis for its anticoagulation activity, we assessed its interactions with various clotting factors. We found that the anticoagulation activity is primarily due to binding to the Gla domains of Factors IX and X, thus preventing these factors from recognizing phosphatidylserine on the plasma membrane. The present study suggests that ligands that bind to the Gla domains of Factors IX and X may have the potential to become novel anticoagulants.