Stochastic Sensing of Nanomolar Inositol 1,4,5-Trisphosphate with an Engineered Pore

AbstractThe introduction of a ring of arginine residues near the constriction in the transmembrane β barrel of the staphylococcal α-hemolysin heptamer yielded a pore that could be almost completely blocked by phosphate anions at pH 7.5. Block did not occur with other oxyanions, including nitrate, sulfate, perchlorate, and citrate. Based on this finding, additional pores were engineered with high affinities for important cell signaling molecules, such as the Ca2+-mobilizing second messenger inositol 1,4,5-trisphosphate (IP3), that contain phosphate groups. One of these engineered pores, PRR-2, provides a ring of fourteen arginines that project into the lumen of the transmembrane barrel. Remarkably, PRR-2 bound IP3 with low nanomolar affinity while failing to bind another second messenger, adenosine 3′, 5′-cyclic monophosphate (cAMP). The engineered α-hemolysin pores may be useful as components of stochastic sensors for cell signaling molecules

High temperature ion channels and pores

The present invention includes an apparatus, system and method for stochastic sensing of an analyte to a protein pore. The protein pore may be an engineer protein pore, such as an ion channel at temperatures above 55.degree. C. and even as high as near 100.degree. C. The analyte may be any reactive analyte, including chemical weapons, environmental toxins and pharmaceuticals. The analyte covalently bonds to the sensor element to produce a detectable electrical current signal. Possible signals include change in electrical current. Detection of the signal allows identification of the analyte and determination of its concentration in a sample solution. Multiple analytes present in the same solution may also be detected

Nanopore-facilitated single molecule detection of circulating microRNAs in cancer patients

Developing new technologies for cancer screening and early diagnosis is a critical issue for saving cancer patients' lives. MicroRNAs (miRNAs) are a class of short (~18-24-nt) non-coding RNAs molecules that regulate gene expression at the post-transcriptional level. Aberrant expression of miRNAs has been found in all types of tumors. Thus miRNAs have been recognized as potential cancer biomarkers. Most notably, specific miRNAs are released from the primary tumor into blood circulation, making the detection of circulating miRNAs profile a powerful tool for noninvasive cancer detection, diagnosis, staging, and monitoring. We developed a robust nanopore sensor that selectively detects single molecules of circulating miRNAs derived from primary cancer. The nanopore is a fabricated 2-nm molecular pore. Such a tiny pore can generate a signature current signal when a miRNA molecule is specifically captured in it. These signals function as fingerprints that enable us to identify a specific miRNA and quantify its concentration. The prototype of nanopore sensor has demonstrated the capability to discriminate single nucleotide difference between miRNAs (single nucleotide polymorphisms, SNPs). In clinical tests, the nanopore has shown the power to differentiate miRNA levels in blood from lung cancer patients and healthy people. Due to the label-free single molecule detection without nucleic acids amplification, the nanopore sensor is higher selective, precise and accurate over the gold standard RT-PCR and microarray. This noninvasive clinical test requires a mere 5 ml of peripheral blood, with a reduced cost from several hundred dollars today to less than 20 dollars per sample. The developing nanopore array would give a high throughput capability for detecting miRNA profile. If validated in clinical trial, the nanopore sensor will become a system available to monitor cancer patients and to screen high risk populations for early diagnosis of cancers which will potentially save the lives of millions. Potential Areas of Applications: * Detection of microRNAs, study of DNA, RNA, DNA/RNA hybrid unzipping kinetics * Study of miRNA mechanism and principle of regulation * Drug discovery and development * Biomarker characterization * Diagnostics and prognostics

Prolonged Residence Time of a Noncovalent Molecular Adapter, β-Cyclodextrin, within the Lumen of Mutant α-Hemolysin Pores

Noncovalent molecular adapters, such as cyclodextrins, act as binding sites for channel blockers when lodged in the lumen of the α-hemolysin (αHL) pore, thereby offering a basis for the detection of a variety of organic molecules with αHL as a sensor element. β-Cyclodextrin (βCD) resides in the wild-type αHL pore for several hundred microseconds. The residence time can be extended to several milliseconds by the manipulation of pH and transmembrane potential. Here, we describe mutant homoheptameric αHL pores that are capable of accommodating βCD for tens of seconds. The mutants were obtained by site-directed mutagenesis at position 113, which is a residue that lies near a constriction in the lumen of the transmembrane β barrel, and fall into two classes. Members of the tight-binding class, M113D, M113N, M113V, M113H, M113F and M113Y, bind βCD ∼10(4)-fold more avidly than the remaining αHL pores, including WT-αHL. The lower K (d) values of these mutants are dominated by reduced values of k(off). The major effect of the mutations is most likely a remodeling of the binding site for βCD in the vicinity of position 113. In addition, there is a smaller voltage-sensitive component of the binding, which is also affected by the residue at 113 and may result from transport of the neutral βCD molecule by electroosmotic flow. The mutant pores for which the dwell time of βCD is prolonged can serve as improved components for stochastic sensors

How to get through a nanopore and across a bilayer: The difference between linear and folded DNA

Abstract only availableTranslocation of DNA through lipid bilayers is essential to cell function for the transcription of RNA and proteins. Alpha hemolysin is the biological nanopore used to translocate DNA across this phospholipid bilayer. There are several variables that affect the translocation of DNA through biological nanopores, these include; cations, applied voltage and the type of DNA being used. For our experiment we are testing the affect of Potassium, Sodium, and Barium cations through a spectrum of 90mV to 180mV on the translocation of two separate types of DNA; Ctrl-2 (linear) and TBA (folding/linear). Through this testing we are trying to determine the correlation between linear DNA (Ctrl-2) and a mixture of folded and linear strands (TBA). Both voltage and cations directly determine the frequency of translocation events of the DNA. If both types of DNA are affected proportionally, we can conclude that the affects of these variables are universal regardless of the differences in structure between the two strands. Preliminary results from the Potassium cation confirm our hypothesis that the results for each DNA (Ctrl-2 and TBA) are very similar with respect to the frequency of translocation events due to the Potassium cation and various voltages. Studies for the Barium and Sodium cations are ongoing.College of Engineering Undergraduate Research Optio

Different yellowing degrees and the industrial utilization of flue-cured tobacco leaves

Yellowing is a key stage in the curing of flue-cured tobacco (Nicotiana tobacum L.) as much of the chemical transformation occurs during this period. This study examined the effect of different yellowing degrees on the value of flue-cured tobacco leaves at the farm level for both processing and manufacturing. The study was conducted in the counties of Chuxiong, Dali, and Yuxi in Yunnan, China over two years. Yellowing treatments have been designed to have either a mild or a regular yellowing degree. Yield, value, appearance, suction property, smoking characteristics, and physical resistance to further processing were investigated to evaluate the effect of degree of yellowing on the industrial utilization of flue-cured tobacco leaves. The regular yellowing degree enhanced yield, value, and appearance compared to the mild yellowing degree, regardless of cultivar or location; however, physical resistance to further processing and the suction property of the mild yellowing degree treatment were better than with the regular yellowing degree regardless of cultivar or location. Furthermore, although the regular yellowing degree recorded higher smoking characteristic scores than the mild yellowing degree immediately after flue-curing, the scores of mild yellowing degree leaves could be further augmented by increasing intensity in the re-drying stage. The smoking characteristic score in the regular yellowing degree can only be increased by low intensity re-drying, and significantly decreased by mild and high intensity re-drying. Therefore, in terms of industrial utilization, mild yellowing is the better choice for flue-curing tobacco. This study also suggested that the current regular yellowing stage in Yunnan should be shortened to meet the demands of the traditional tobacco industry