Hydrogen terminated silicon surfaces: Development of sensors to detect metallic contaminants and stability studies under different environments

Hydrogen terminated silicon surfaces have been utilized to develop sensors for semiconductor and environmental applications. The interaction of these surfaces with different environments has also been studied in detail. The sensor assembly relevant to the semiconductor industry utilizes a silicon-based sensor to detect trace levels of metallic contaminants in hydrofluoric acid. The sensor performance with respect to two non-contaminating reference electrode systems was evaluated. In the first case, conductive diamond was used as a reference electrode. In the second case, a dual silicon electrode system was used with one of the silicon-based electrodes protected with an anion permeable membrane behaving as the quasi reference electrode. Though both systems could function well as a suitable reference system, the dual silicon electrode design showed greater compatibility for the on-line detection of metallic impurities in HF etching baths. The silicon-based sensor assembly was able to detect parts- per-trillion to parts-per-billion levels of metal ion impurities in HF. The sensor assembly developed for the environmental application makes use of a novel method for the detection of Ni2+using attenuated total reflection (ATR) technique. The nickel infrared sensor was prepared on a silicon ATR crystal uniformly coated by a 1.5 micron Nafion film embedded with dimethylglyoxime (DMG) probe molecules. The detection of Ni2+ was based on the appearance of a unique infrared absorption peak at 1572 cm-1 that corresponds to the C=N stretching mode in the nickel dimethylglyoximate, Ni(DMG)2, complex. The suitable operational pH range for the nickel infrared sensor is between 6-8. The detection limit of the nickel infrared sensor is 1 ppm in the sample solution of pH=8. ATR - FTIR spectroscopy was used to study the changes that the hydride mode underwent when subjected to different environments. The presence of trace amounts of Cu2+ in HF solutions was found to roughen the silicon surface as observed ATR-IR spectroscopy. The initial stages of oxidation in UPW and Cu2+ / UPW was studied. Trace amounts of Cu2+ were found to drastically increase the rate of oxidation, while the rate of oxidation was found to be retarded on removing dissolved oxygen that was present in UPW

Role of Polypyrimidine Tract Binding Protein in Mediating Internal Initiation of Translation of Interferon Regulatory Factor 2 RNA

BACKGROUND: Earlier we have reported translational control of interferon regulatory factor 2 (IRF2) by internal initiation (Dhar et al, Nucleic Acids Res, 2007). The results implied possible role of IRF2 in controlling the intricate balance of cellular gene expression under stress conditions in general. Here we have investigated the secondary structure of the Internal Ribosome Entry Site of IRF2 RNA and demonstrated the role of PTB protein in ribosome assembly to facilitate internal initiation. METHODOLOGY/PRINCIPAL FINDINGS: We have probed the putative secondary structure of the IRF2 5'UTR RNA using various enzymatic and chemical modification agents to constrain the secondary structure predicted from RNA folding algorithm Mfold. The IRES activity was found to be influenced by the interaction of trans-acting factor, polypyrimidine tract binding protein (PTB). Deletion of 25 nts from the 3'terminus of the 5'untranslated region resulted in reduced binding with PTB protein and also showed significant decrease in IRES activity compared to the wild type. We have also demonstrated putative contact points of PTB on the IRF2-5'UTR using primer extension inhibition assay. Majority of the PTB toe-prints were found to be restricted to the 3'end of the IRES. Additionally, Circular Dichroism (CD) spectra analysis suggested change in the conformation of the RNA upon PTB binding. Further, binding studies using S10 extract from HeLa cells, partially silenced for PTB gene expression, resulted in reduced binding by other trans-acting factors. Finally, we have demonstrated that addition of recombinant PTB enhances ribosome assembly on IRF2 IRES suggesting possible role of PTB in mediating internal initiation of translation of IRF2 RNA. CONCLUSION/SIGNIFICANCE: It appears that PTB binding to multiple sites within IRF2 5'UTR leads to a conformational change in the RNA that facilitate binding of other trans-acting factors to mediate internal initiation of translation

Cryptic AUG is important for 48S ribosomal assembly during internal initiation of translation of coxsackievirus B3 RNA

We have investigated the possible role of a conserved cis-acting element, the cryptic AUG, present in the 5' UTR of coxsackievirus B3 (CVB3) RNA. CVB3 5' UTR contains multiple AUG codons upstream of the initiator AUG, which are not used for the initiation of translation. The 48S ribosomal assembly takes place upstream of the cryptic AUG. We show here that mutation in the cryptic AUG results in reduced efficiency of translation mediated by the CVB3 IRES; mutation also reduces the interaction of mutant IRES with a well characterized IRES trans-acting factor, the human La protein. Furthermore, partial silencing of the La gene showed a decrease in IRES activity in the case of both the wild-type and mutant. We have demonstrated here that the interaction of the 48S ribosomal complex with mutant RNA was weaker compared with wild-type RNA by ribosome assembly analysis. We have also investigated by chemical and enzymic modifications the possible alteration in secondary structure in the mutant RNA. Results suggest that the secondary structure of mutant RNA was only marginally altered. Additionally, we have demonstrated by generating compensatory and non-specific mutations the specific function of the cryptic AUG in internal initiation. Results suggest that the effect of the cryptic AUG is specific and translation could not be rescued. However, a possibility of tertiary interaction of the cryptic AUG with other cis-acting elements cannot be ruled out. Taken together, it appears that the integrity of the cryptic AUG is important for efficient translation initiation by the CVB3 IRES RNA

Effect of mutations on the p53 IRES RNA structure Implications for de-regulation of the synthesis of p53 isoforms

Earlier we have demonstrated the presence of internal ribosome entry site (IRES) within tumor suppressor p53 mRNA. Here we have mapped the putative secondary structure of p53-IRES RNA using information from chemical probing and nuclease mapping experiments. Additionally, the secondary structure of the IRES element of the wild-type RNA was compared with cancer-derived silent mutant p53 RNAs. These mutations might result in the conformational alterations of p53-IRES RNAs. The results also indicate decreased IRES activities of the mutants as compared to wild-type RNA. Further, it was observed that some of the cytoplasmic trans-acting factors, critical for enhancing IRES function, were unable to bind mutant RNAs as efficiently as to wild-type. Our results suggest that hnRNP C1/C2 binds to p53-IRES and siRNA mediated partial silencing of hnRNP C1/C2 showed appreciable decrease in IRES function and consequent decrease in the level of the corresponding p53 isoform. Interestingly mutant p53 IRES showed lesser binding with hnRNP C1/C2 protein. Finally, upon doxorubicin treatment, the mutant RNAs were unable to show enhanced p53 synthesis to similar extent compared to wild type. Taken together, these observations suggest that mutations occurring in the p53 IRES might have profound implications for de-regulation of its expression and activity

Probing of secondary structure of IRF2 UTR RNA with DMS and RNase T1:

<p>(A) A schematic representation of IRF2-Luc monocistronic construct used for generating <i>in vitro</i> transcripts by using T7 RNA polymerase. A 20 nucleotide long -³²P labeled reverse primer was used for reverse transcription indicated by an arrow at the 5′ end of luciferase. (B) IRF2 5′ UTR RNA was incubated with (lane 2) or without (lane 1) dimethyl sulphate (DMS). The unmodified and modified RNAs were reverse transcribed and cDNAs were resolved parallel with a sequencing reaction performed with the same end labeled primer. Modified nucleotides are indicated on the right of the panel. (C) IRF2 5′ UTR RNA was incubated with or without of 1.0 unit of RNase T1 and the undigested and digested RNAs were reverse transcribed and cDNAs were resolved in parallel with a sequencing reaction performed with the same end labeled primer. Putative cleavage points are indicated by arrows on the right of the panel.</p

Analysis of translation initiation complex formation in presence of recombinant PTB:

<p>³²P UTP labeled IRF2 UTR was incubated with RRL and amino acid mix and analyzed by sucrose density gradients. Panel A represents the control. Panel B shows the profile of the respective reactions supplemented with either 1.75 or 3.5 nM of purified recombinant PTB protein (as indicated). The panels C–D shows the effect of addition of either GMPPNP (panel C) or Cycloheximide (panel D) used to confirm the 48S and 80S ribosomal peaks (indicated by arrows).</p

Schematic diagram MFOLD structure of IRF2 5′UTR:

<p>Predicted secondary structure of IRF2 5′UTR generated by MFOLD indicating putative contact points of PTB, DMS modification, RNase V1 or T1 cleavage sites.</p

PTB protein helps interaction of other transacting factors with the IRF2-IRES:

<p>(A) ³²P labeled IRF2 WT 5′UTR and mIRF2 5′UTR were crosslinked with total HeLa S10 extracts and run on an 10% SDS PAGE followed by phosphorimaging. Lane 1 represents no protein control. Lane M indicates the molecular mass marker. (B) 70% confluent HeLa cells were transfected with 60 nm and 80 nm of siPTB or nsp si as indicated. 36 hrs post-transfection the cells were harvested and subjected for western blot analysis to check PTB levels. S10 extract was prepared from these cells and UV Crosslinking was performed as mentioned previously. Lane M indicates the molecular mass marker. (C) Western-blot analysis showing the reduced levels of PTB with increasing concentration of siPTB. Actin protein was detected as loading control.</p

RNase VI digestion and Toe-printing of PTB on IRF2 UTR RNA:

<p>(A) IRF2 5′ UTR RNA was incubated with (lane 2) or without (lane 1) of 0.01 units of RNase V1. The unmodified and modified RNAs were reverse transcribed and cDNAs were resolved parallel with a sequencing reaction performed with the same end labeled primer. Cleaved nucleotides are indicated on the right of the panel. (B) For toe-printing, IRF2 wt 5′ UTR RNA was incubated in absence or presence of purified recombinant PTB protein. The RNAs in the Ribonucleoprotein complexes were reverse transcribed and the cDNAs were resolved in 8M urea 8% PAGE in parallel with a sequencing ladder corresponding to IRF2 5′UTR RNA obtained by using the same end labeled primer. The cDNA products terminated at the sites due to protein binding is indicated on the right.</p