Electron Transport in Muscle Protein Collagen.

In recent times, collagen, which is one of the most abundant proteins in animals, has appeared to be an attractive candidate for biomaterial applications, for example, in medical implants and wearable electronics. This is because collagen is water-insoluble, biocompatible, and nontoxic. In addition, films of different sizes and shapes can be made using this protein as it is malleable and elastic in nature. However, its electron transport capacity or its absence has remained largely untested so far. Therefore, in this work, the electron transport behavior of collagen has been studied in both film and single-fiber states in a local probe configuration using current-sensing atomic force spectroscopy (CSAFS). From the CSAFS analyses, the electronic (transport) band gap of collagen has been estimated. It has been found that collagen behaves as a wide band gap semiconductor (near-insulating) in a variety of experimental conditions. The transition to a semiconducting material with a low electronic band gap and a nearly 1000-fold enhancement of current (picoampere to nanoampere level) occurs by metal ion treatment (here, Fe3+) of the native collagen. To the best of our knowledge, this is the first report of a molecular level study of the electron transport behavior of collagen proteins and estimation of transport band gap values of collagen and metalated collagen.

Nanoscale Nucleic Acid Recognition at the Solid-Liquid Interface Using Xeno Nucleic Acid Probes.

Challenges in reliable nucleic acid detection are manifold. The major ones are related to false positive or negative signals due to a lack of target specificity in detection and to low sensitivity, especially when a plethora of background sequences are present that can mask the specific recognition signal. Utilizing designed synthetic nucleic acids that are commonly called xeno nucleic acids could offer potential routes to meeting such challenges. In this article, we present the general framework of nucleic acid detection, especially for nanoscale applications, and discuss how and why the xeno nucleic acids could be truly an alternative to the DNA probes. Two specific cases, locked nucleic acid (LNA) and peptide nucleic acid (PNA), which are nuclease-resistant and can form thermally stable duplexes with DNA, are addressed. It is shown that the relative ease of the conformationally rigid LNA probe to be oriented upright on the substrate surface and of the nonionic PNA probe to result into high probe density assists in their use in nanoscale nucleic acid recognition. It is anticipated that success with these probes may lead to important developments such as PCR-independent approaches where the major aim is to detect a small number of target sequences present in the analyte medium.

Molecularly resolved label-free sensing of single nucleobase mismatches by interfacial LNA probes.

So far, there has been no report on molecularly resolved discrimination of single nucleobase mismatches using surface-confined single stranded locked nucleic acid (ssLNA) probes. Herein, it is exemplified using a label-independent force-sensing approach that an optimal coverage of 12-mer ssLNA sensor probes formed onto gold(111) surface allows recognition of ssDNA targets with twice stronger force sensitivity than 12-mer ssDNA sensor probes. The force distributions are reproducible and the molecule-by-molecule force measurements are largely in agreement with ensemble on-surface melting temperature data. Importantly, the molecularly resolved detection is responsive to the presence of single nucleobase mismatches in target sequences. Since the labelling steps can be eliminated from protocol, and each force-based detection event occurs within milliseconds' time scale, the force-sensing assay is potentially capable of rapid detection. The LNA probe performance is indicative of versatility in terms of substrate choice - be it gold (for basic research and array-based applications) or silicon (for 'lab-on-a-chip' type devices). The nucleic acid microarray technologies could therefore be generally benefited by adopting the LNA films, in place of DNA. Since LNA is nuclease-resistant, unlike DNA, and the LNA-based assay is sensitive to single nucleobase mismatches, the possibilities for label-free in vitro rapid diagnostics based on the LNA probes may be explored.

Nanoscale On-Silico Electron Transport via Ferritins.

Silicon is a solid-state semiconducting material that has long been recognized as a technologically useful one, especially in electronics industry. However, its application in the next-generation metalloprotein-based electronics approaches has been limited. In this work, the applicability of silicon as a solid support for anchoring the iron-storage protein ferritin, which has a semiconducting iron nanocore, and probing electron transport via the ferritin molecules trapped between silicon substrate and a conductive scanning probe has been investigated. Ferritin protein is an attractive bioelectronic material because its size (X-ray crystallographic diameter ∼12 nm) should allow it to fit well in the larger tunnel gaps (>5 nm), fabrication of which is relatively more established, than the smaller ones. The electron transport events occurring through the ferritin molecules that are covalently anchored onto the MPTMS-modified silicon surface could be detected at the molecular level by current-sensing atomic force spectroscopy (CSAFS). Importantly, the distinct electronic signatures of the metal types (i.e., Fe, Mn, Ni, and Au) within the ferritin nanocore could be distinguished from each other using the transport band gap analyses. The CSAFS measurements on holoferritin, apoferritin, and the metal core reconstituted ferritins reveal that some of these ferritins behave like n-type semiconductors, while the others behave as p-type semiconductors. The band gaps for the different ferritins are found to be within 0.8 to 2.6 eV, a range that is valid for the standard semiconductor technology (e.g., diodes based on p-n junction). The present work indicates effective on-silico integration of the ferritin protein, as it remains functionally viable after silicon binding and its electron transport activities can be detected. Potential use of the ferritin-silicon nanohybrids may therefore be envisaged in applications other than bioelectronics, too, as ferritin is a versatile nanocore-containing biomaterial (for storage/transport of metals and drugs) and silicon can be a versatile nanoscale solid support (for its biocompatible nature).

Discriminating unalike single nucleobase mismatches using a molecularly resolved, label-free, interfacial LNA-based assay.

A number of reports have been made in recent times on label-free detection of nucleic acid sequences. However, most of these studies deal with ensemble measurements, therefore lacking in molecular level resolution. These assays have usually employed ssDNA sensor probes, and often suffered from problems of irreproducibility and poor sequence-selectivity. Herein, the applicability of surface-anchored single stranded locked nucleic acid (ssLNA) probes has been assessed in the detection of target DNA sequences, as an alternative to the DNA-based assay. Importantly, the effectiveness of the LNA-based assay in identifying different types of single nucleobase mismatches has been tested. Since the duplex melting temperature is an indicator of duplex stability, the ensemble on-surface Tm values of the surface-confined LNA-DNA duplexes have been compared to the duplex unbinding force values obtained from atomic force spectroscopy (AFS) experiments. A common mismatch discrimination pattern elicited by both the ensemble and the molecular level AFS approach could be identified. Apart from quantitative delineation of the different types of mismatches, the label-free AFS analysis confirms different degrees of efficiency of the purine and pyrimidine bases, present on the LNA backbone, in discriminating different nucleobase mismatch types. Importantly, the LNA-based AFS analysis can distinguish between the disease-relevant gene fragments, e.g., multidrug-resistant Mycobacterium tuberculosis (MTB) mutation, and the wild type. Since LNA probes are nuclease-resistant, these findings could potentially pave way to diagnostic applications of the LNA-based AFS assay.

Regulating the on-surface LNA probe density for the highest target recognition efficiency.

The recent emergence of on-surface LNA-based assays as potentially better alternatives over DNA-based approaches, due to enhanced sensitivity and target specificity, raises the need for the precise identification of the factors that control the performance of these assays. In this work, we investigated whether the probe density of fully modified ssLNA probes on the gold(111) surface could influence the target recognition capacity of the LNA sensing layer and illustrated simple means to control it, primarily by adjusting the salt concentration, nature of the cation, and pH of the immobilization buffer. It was observed that monovalent Na(+) could more effectively control the sensor probe density compared to bivalent Mg(2+), leading to better target recognition. Interestingly, unlike in the case of ssDNA sensor probes, the target recognition efficiency of the LNA layer at the optimum probe density was found to be almost spacer-independent, probably due to the rigidity of the LNA backbone. The optimized LNA sensor layer could discriminate single base mismatches, detect a minimum target DNA concentration of 5 nM, and sense a significant level of hybridization within a time scale of a few minutes. To our knowledge, for the first time, we identify the factors that control the on-surface LNA probe density for maximizing the performance of the LNA sensing layer.

Molecular Crosstalk Between Adherens Junction Proteins, E-cadherin and Nectin-4.

Cell-cell junctions formed by the association of cell adhesion molecules facilitate physiological events necessary for growth and development of multicellular organisms. Among them, cadherins and nectins organize and assemble to form adherens junction, which thereby mechanically couples interacting cells. A detailed understanding of the crosstalk involving these cell adhesion molecules is fundamental to the study of the various developmental processes. Although, cadherins and nectins can recruit each other in the adherens junction through an interplay of cytoplasmic adaptor molecules, here, we report a direct interaction between N-terminal extracellular domains of E-cadherin and nectin-4 as demonstrated by surface plasmon resonance (SPR) and Atomic Force Microscopy (AFM)-based single molecule force spectroscopy (SMFS). Kinetic studies using SPR demonstrate the binding between the ectodomains of E-cadherin and nectin-4 with a KD of 3.7 ± 0.7 µM and KD of 5.4 ± 0.2 µM (reciprocal experiment). AFM-based SMFS experiments also support interaction between the ectodomains of E-cadherin and nectin-4 with the koff value of 31.48 ± 1.53 s-1 and the lifetime of the complex of 0.036 ± 0.0026 s. We thus propose a cell adhesion mechanism mediated by E-cadherin and nectin-4, which can have functional significance in early embryogenesis as evident from the expression pattern of both the proteins during early development.

Maximizing mismatch discrimination by surface-tethered locked nucleic acid probes via ionic tuning.

Several investigations on DNA-based nucleic acid sensors performed in the past few years point toward the requirement of an alternative nucleic acid that can detect target DNA strands more efficiently, i.e., with higher sensitivity and selectivity, and can be more robust compared to the DNA sensor probes. Locked nucleic acid (LNA), a conformationally restricted DNA analogue, is potentially a better alternative than DNA, since it is nuclease-resistant, it can form a more stable duplex with DNA in a sequence-specific manner, and it interacts less with substrate surface due to presence of a rigid backbone. In this work, we probed solid-phase dehybridization of ssDNA targets from densely packed fully modified ssLNA probes immobilized onto a gold(111) surface by fluorescence-based measurement of the "on-surface" melting temperatures. We find that mismatch discrimination can be clearly improved by applying the surface-tethered LNA probes, in comparison to the corresponding DNA probes. We show that concentration as well as type of cation (monovalent and polyvalent) can significantly influence thermal stability of the surface-confined LNA-DNA duplexes, the nature of concentration dependence contradicting the solution phase behavior. Since the ionic setting influenced the fully matched duplexes more strongly than the singly mismatched duplexes, the mismatch discrimination ability of the surface-confined LNA probes could be controlled by ionic modulations. To our knowledge, this is the first report on ionic regulation of melting behavior of surface-confined LNA-DNA duplexes.

Enhancing on-surface mismatch discrimination capability of PNA probes by AuNP modification of gold(111) surface.

Unambiguous identification of single base mismatches in nucleic acid sequences is of great importance in nucleic acid detection assays. However, ambiguities are often encountered with, and therefore, a strategy for attaining substantially large enhancement of mismatch discrimination has been worked upon in this study. Short single-stranded peptide nucleic acid (PNA) and deoxyribonucleic acid (DNA) sensor probes that are immobilized onto gold nanoparticle (AuNP) modified Au(111) surface have been applied for target DNA detection. It will be shown that while both PNA and the analogous DNA probes exhibit generally better target detection abilities on the AuNP-modified Au(111) surface (elicited from fluorescence-based measurement of on-surface Tm values), compared to the bare Au(111) surface, PNA supersedes DNA, for all sizes of AuNPs (10, 50, and 90 nm) applied, with the difference being quite drastic in the case of the smallest 10 nm AuNP. It is found that while the AuNP curvature plays a pivotal role in target detection abilities of the PNA probes, the changes in the surface roughness caused by AuNP treatment do not exert any significant influence. This study also presents a means for preparing PNA-AuNP hybrids without altering PNA functionality and without AuNP aggregation by working with the surface-affixed AuNPs.

Facilitating mismatch discrimination by surface-affixed PNA probes via ionic regulation.

There has been a search for alternative nucleic acids that can be more effectively used in nucleic acid detection technologies compared to the DNA probes. Peptide nucleic acid (PNA), which contains a non-ionic peptidic backbone, offers such possibilities since it is nuclease-resistant, it binds to DNA with high affinity, and it can be readily self-assembled onto solid substrates, e.g., gold(111), with a molecular backbone orientation away from the substrate. Although application of PNA as a sensor probe has been exemplified, so far there is little or no account of the ionic modulation of single base mismatch discrimination capacity of surface-tethered PNA probes. Herein, we report "on-surface" melting temperatures of PNA-DNA duplexes formed on gold(111) surface, as obtained from fluorescence measurements. We show that surface-tethered PNA forms a stabler duplex than DNA, and is more effective in single base mismatch discrimination than DNA. Importantly, although PNA backbone is non-ionic, variation in the ionic components in hybridization buffer, i.e., varying concentration of monovalent sodium ion, and the nature of anion and the cation, exhibits clear effects on the mismatch discrimination capacity of PNA probes. In general, with decreasing cation concentration, PNA-DNA duplexes are stabilized and mismatch discrimination capacity of the PNA probes is enhanced. The stabilizing/destabilizing effects of anions are found to follow the Hofmeister series, emphasizing the importance of hydrophobic interaction between nucleobases for stability of the PNA-DNA duplexes. Interestingly, the nature of ionic dependence of "on-surface" mismatch detection ability of PNA probes differs significantly from the "solution" behavior of these probes.

Nanoscale mechano-electronic behavior of a metalloprotein as a variable of metal content.

In this work, we have explored an approach to finding a correlation between the mechanical response of a metalloprotein against a range of applied force (by force curve analysis) and its electrical response under pressure stimulation (by current sensing atomic force spectroscopy) at the nanoscale. Iron-storage protein ferritin has been chosen as an experimental model system because it naturally contains a semiconducting iron core. This core consists of a large number of iron atoms and is therefore expected to exert a clear influence on the overall mechanical response of the protein structure. Four different ferritins (apoferritin, Fe(III)-ferritins containing ~750 and ~1400 iron atoms, and holoferritin containing ~2600 iron atoms) were chosen in order to identify any relation between the mechano-electronic behavior of the ferritins and their metal content. We report the measurement of Young's modulus values of the ferritin proteins as applicable in a nanoscale environment, for the first time, and show that these values are directly linked to the iron content of the individual ferritin type. The greater the iron content, the greater the Young's modulus and in general the slower the rate of deformation against the application of force. When compressed, all the four ferritins exhibited increased electronic conductivity. A correlation between the iron content of the ferritins and the current values observed at certain bias voltages could be made at higher bias values (beyond 0.7 V), but no such discrimination among the four compressed ferritins could be made at the lower voltages. We propose that only at higher voltages can the iron atoms that reside deeper inside the core of the ferritins be accessed. The iron atoms that could be situated at the inner wall of the protein shell appear to make a general contribution to the electronic conductivity of the four ferritin systems.

Impact of HMGB1 binding on the structural alterations of platinum drug-treated single dsDNA molecule.

High mobility group B1 (HMGB1) is an architectural protein that recognizes the DNA damage sites formed by the platinum anticancer drugs. However, the impact of HMGB1 binding on the structural alterations of the platinum drug-treated single dsDNA molecules has remained largely unknown. Herein, the structural alterations induced by the platinum drugs, the mononuclear cisplatin and it's analog the trinuclear BBR3464, have been probed in presence of HMGB1, by atomic force microscopy (AFM) and AFM-based force spectroscopy. It is observed that the drug-induced DNA loop formation enhanced upon HMGB1 binding, most likely as a result of HMGB1-induced increase in DNA conformational flexibility that allowed the drug-binding sites to come close and form double adducts, thereby resulting in enhanced loop formation via inter-helix cross-linking. Since HMGB1 enhances DNA flexibility, the near-reversible structural transitions as observed in the force-extension curves (for 1 h drug treatment), generally occurred at lower forces in presence of HMGB1. The DNA structural integrity was largely lost after 24 h drug treatment as no reversible transition could be observed. The Young's modulus of the dsDNA molecules, as estimated from the force-extension analysis, increased upon drug treatment, due to formation of the drug-induced covalent cross-links and consequent reduction in DNA flexibility. The Young's modulus increased further in presence of HMGB1 due to HMGB1-induced enhancement in DNA flexibility that could ease formation of the drug-induced covalent cross-links. To our knowledge, this is the first report that shows an increase in the stiffness of the platinum drug-treated DNA molecules in presence of HMGB1.

Ordered self-assembled locked nucleic acid (LNA) structures on gold(111) surface with enhanced single base mismatch recognition capability.

Locked nucleic acid (LNA) is a conformationally restricted nucleic acid analogue, which is potentially a better alternative than DNA for application in the nucleic acid based biosensor technologies, due to its efficient and sequence-specific DNA/RNA detection capability and lack of molecule-surface interaction on solid surfaces, compared to DNA. We report, for the first time, a straightforward way (based on simple immersion method) of generating an ordered self-assembled LNA monolayer, which is bioactive, onto a gold(111) surface. This layer is capable of giving rise to a stronger DNA recognition signal (4-4.5 times) than its DNA counterpart, and importantly, it can differentiate between a fully complementary DNA target and that having a single base mismatch, where the mismatch discrimination ratio is almost two times compared to the ratio relevant in case of DNA-based detection. We have presented high-resolution atomic force microscopy (AFM) topographs of the well-defined one-dimensional LNA molecular ordering (few hundred nanometers long) and of the two-dimensional ordered assembly formed over a large area (7 µm × 7 µm) due to parallel positioning of the one-dimensional ordered arrangements. The effects of different parameters such as LNA concentration and incubation time on LNA self-assembly have been investigated. Further, reflection absorption infrared (RAIR) spectroscopy has been applied to obtain information about the orientation of the surface-immobilized LNA molecules for the first time. It has been found that the LNA molecules undergo an orientational transition from the "lying down" to the "upright" configuration in a time scale of few hours.

XNAs: A Troubleshooter for Nucleic Acid Sensing.

The strategies for nucleic acid sensing based on nucleic acid hybridization between the target sequence and the capture probe sequence are considered to be largely successful as far as detection of a specific target of known sequence is concerned. However, when compared with other complementary methods, like direct sequencing, a number of results are still found to be either "false positives" or "false negatives". This suggests that modifications in these strategies are necessary to make them more accurate. In this minireview, we propose that one way toward improvement could be replacement of the DNA capture probes with the xeno nucleic acid or XNA capture probes. This is because the XNAs, especially the locked nucleic acid, the peptide nucleic acid, and the morpholino, have shown better single nucleobase mismatch discrimination capacity than the DNA capture probes, indicating their capacity for more precise detection of nucleic acid sequences, which is beneficial for detection of gene stretches having point mutations. Keeping the current trend in mind, this minireview will include the recent developments in nanoscale, fluorescent label-free applications, and present the cases where the XNA probes show clear advantages over the DNA probes.

Molecularly resolved, label-free nucleic acid sensing at solid-liquid interface using non-ionic DNA analogues.

Nucleic acid-based biosensors, where the capture probe is a nucleic acid, e.g., DNA or its synthetic analogue xeno nucleic acid (XNA), offer interesting ways of eliciting clinically relevant information from hybridization/dehybridization signals. In this respect, the application of XNA probes is attractive since the drawbacks of DNA probes might be overcome. Within the XNA probe repertoire, peptide nucleic acid (PNA) and morpholino (MO) are promising since their backbones are non-ionic. Therefore, in the absence of electrostatic charge repulsion between the capture probe and the target nucleic acid, a stable duplex can be formed. In addition, these are nuclease-resistant probes. Herein, we have tested the molecularly resolved nucleic acid sensing capacity of PNA and MO capture probes using a fluorescent label-free single molecule force spectroscopy approach. As far as single nucleobase mismatch discrimination is concerned, both PNA and MO performed better than DNA, while the performance of the MO probe was the best. We propose that the conformationally more rigid backbone of MO, compared to the conformationally flexible PNA, is an advantage for MO, since the probe orientation can be made more upright on the surface and therefore MO can be more effectively accessed by the target sequences. The performance of the XNA probes has been compared to that of the DNA probe, using fixed nucleobase sequences, so that the effect of backbone variation could be investigated. To our knowledge, this is the first report on molecularly resolved nucleic acid sensing by non-ionic capture probes, here, MO and PNA.

Tuning band gap of holoferritin by metal core reconstitution with Cu, Co, and Mn.

Utility of ferritin in molecular electronics, especially in single molecule electronics based devices, has recently been proposed, since the iron core of holoferritin is semiconducting in nature. However, the practical aspects, e.g., how its electronic properties can be varied/tuned, need to be better addressed. In this direction, we have performed direct tunneling experiments using scanning tunneling microscopy (STM) and scanning tunneling spectroscopy (STS) on several metal core reconstituted ferritins, where the reconstitution has been carried out using biocompatible metals like copper, cobalt, and manganese that are found naturally in the human body. We show, for the first time, that, by metal core reconstitution of the ferritin protein, the band gap of the protein can be tuned to different values (here, within the range 1.17-0.00 eV, considering iron-containing holoferritin and apoferritin as well). From the respective current-voltage curves and the well-defined band gaps, clear distinction can be made among the five different ferritins indicating that the metal core has direct contribution in the observed electrical conductivities of ferritins. It is further revealed that the electrical conductivities of the reconstituted ferritins are of the same order as that for the free metal conductivities, meaning that the relative changes in the free metal conductivities are reflected in the contributions of the metals in protein shell-confinement (i.e., the ∼8 nm core of ferritin). This finding could lead to a strategy for fine-tuning ferritin band gap by preselecting a metal on the basis of the free metal conductivity values.

How stable are the collagen and ferritin proteins for application in bioelectronics?

One major obstacle in development of biomolecular electronics is the loss of function of biomolecules upon their surface-integration and storage. Although a number of reports on solid-state electron transport capacity of proteins have been made, no study on whether their functional integrity is preserved upon surface-confinement and storage over a long period of time (few months) has been reported. We have investigated two specific cases-collagen and ferritin proteins, since these proteins exhibit considerable potential as bioelectronic materials as we reported earlier. Since one of the major factors for protein degradation is the proteolytic action of protease, such studies were made under the action of protease, which was either added deliberately or perceived to have entered in the reaction vial from ambient environment. Since no significant change in the structural characteristics of these proteins took place, as observed in the circular dichroism and UV-visible spectrophotometry experiments, and the electron transport capacity was largely retained even upon direct protease exposure as revealed from the current sensing atomic force spectroscopy experiments, we propose that stable films can be formed using the collagen and ferritin proteins. The observed protease-resistance and robust nature of these two proteins support their potential application in bioelectronics.

Free-Energy-Based Gene Mutation Detection Using LNA Probes.

We have developed a label-free approach for direct detection of gene mutations using free-energy values that are derived from single-molecule force spectroscopy (SMFS)-based nucleic acid unbinding experiments. From the duplex unbinding force values acquired by SMFS, the force-loading-rate-independent Gibbs free-energy values were derived using Jarzinsky's equality treatment. Because it provides molecule-by-molecule information, this approach is a major shift compared to the earlier reports on label-free detection of DNA sequences, which are mostly based on ensemble level data. We tested our approach in the disease model framework of multiple drug-resistant tuberculosis using the nuclease-resistant and conformationally rigid locked nucleic acid probes that are a robust and efficient alternative to the DNA probes. All of the major mutations in Mycobacterium tuberculosis (MTB), as relevant to MTB's resistance to the first-line anti-TB drugs rifampicin and isoniazid, could be identified, and the wild type could be discriminated from the most prevalent mutation and the most prevalent mutation from the less occurring ones. Our approach could also identify DNA sequences (45 mer), having overhang stretches at different positions with respect to the complementary stretch. Probably for the first time, the findings show that free-energy-based detection of gene mutations is possible at molecular resolution.

A new bio-active surface for protein immobilisation via copper-free 'click' between azido SAM and alkynyl Fischer carbene complex.

Fischer carbene complex anchored on glass or silicon surface using a Cu-free 'click' reaction allows facile and swift covalent grafting of protein molecules like Bovine Serum Albumin (BSA).

Polymorphic CUG repeats in human mRNAs and their effects on gene expression.

Expanded CUG repeats in the 3'-untranslated region (UTR) of the gene encoding myotonic dystrophy protein kinase (DMPK) cause myotonic dystrophy type 1 disease (DM1). The presence of such repeats has been found to impede gene expression at several levels in model systems. We took a bioinformatic approach to survey all human mRNA sequences for polymorphic CUG repeats. Our survey revealed that CUG repeats occur widely in various regions of mRNAs, with higher frequency in protein coding regions than 5'-UTRs or 3'-UTRs. About 30 genes were found to contain CUG repeats that are polymorphic in the number of repeats, suggesting the potential to expand or shrink. However, long polymorphic repeats were restricted to the 3'-UTR of the DMPK gene and the coding region of the ribosomal protein L14 gene. Using cell-free translation systems, we showed that extended CUG repeats can inhibit protein synthesis in vitro in the rabbit reticulocyte lysate, but not in wheat germ extracts, consistent with our previous finding of an interaction of CUG repeats with the protein kinase PKR. In transfected cells, CUG repeats can inhibit gene expression both in cis and in trans. However, observations with PKR-minus cells indicate that these effects are not primarily attributable to the interaction of extended CUG repeats with PKR. Northwestern blotting detected the presence in human cells of more CUG-binding proteins than are currently known.