This thesis focuses on making thermodynamic nucleicaciddesign a high-quality, versatile, and low-cost technology that is easy to use and easily accessible. We demonstrated how to achieve high-quality sequences via ensemble defect optimization for only 4/3 the cost of the objective function for large structures. We have further increased design versatility by developing an algorithm for the design of multi-state systems of nucleic acids that also achieves high-quality sequence for low cost with ensemble defect optimization. Finally, we have presented a web server that makes parallel implementations of these and other algorithms easy to use and accessible with the use of a specialized front-end to a high-performance compute cluster. The highly interactive features and visualization tools of NUPACK allow for users to explore and communicate their results with greater understanding and further reach than was previously possible. Nevertheless, improving cost, scope, and availability remains an open area of research.
design forecasts that both PNA and PCR primer target sites overlap, thus leading to a direct competition towards complementary DNA (Fig. 1). When perfect matching occurs PNA-template hybridization is favoured more than primer template duplex and DNA amplifica- tion is suppressed. Conversely, a single mismatch destabilizes the PNA-template duplex, favouring the hybridization between template and primer thus allowing template amplification. Competitor PNA sequence was designed to perfectly match wild-type (WT) template se- quence. Therefore, when a single base pair mismatch oc- curs (like in the case of T315I) PNA-template stability is strongly impaired and DNA amplification favoured.
We thank Denise Martin, Alison Johnson, and Jason Velez for serological characterization of the human CSF samples used in this project; Roger Nasci, Marvin Godsey, Carl Mitchell, Harry Savage, Nicholas Komar, Nicholas Panella, Kristy Gottfried, and Chris Happ for mosquito pool and avian tissue preparation and Vero cell culture assay; Grant Campbell (CDC) and Marci Layton, Annie Fine, Dennis Nash, Alex Ramon, and Iqbal Poshni (all New York City Department of Health) for providing human specimens for testing; Brian Holloway (CDC) and the staff at the CDC Scientific Resources Program for assistance in designing the TaqMan assay primers and probes and for oligonucleotide synthesis; and Pierre van Aarle, Birgit Deiman, Lynell Grosso, and Mike Cronin (bioMe´rieux) for assistance in designing the NASBA-beacon assay probes and NASBA assay design.
Performance parameters for each approach were ana- lyzed by the MedCalc ® 15.2.2 and SPSS22.0. Positive results from patients with proven or probable IA accord- ing to the EORTC/MSG criteria were considered to be true positives and used to calculate the sensitivity of the three assays. For calculation of specificity, negative results from patients without EORTC/MSG evidence of IA were considered to be true negatives. Receiver operating char- acteristic curve (ROC) was used to assess the diagnostic value of NASBA-ELISA system. A higher value of area under the curve (AUC) represents a greater diagnostic value of the assay. Comparison between two methods was performed by paired diagnosis test design using Fisher’s exact test to generate two-sided P values with a P value of ≤0.05 being considered significant. Absorb- ance readings and arithmetical means of the number of Aspergillus spores (transformed into log scale) were ana- lyzed by Pearson’s parametric correlation coefficient or Spearman’s nonparametric correlation coefficient in the NASBA-ELISA test. The Youden index was calculated to evaluate the synthetic ability of each assay. A kappa statistic was determined and interpreted as described previously. Values greater than 0.8 means excellent agree- ment between methods, values of 0.61–0.8 means sub- stantial agreement, values of 0.41–0.6 means moderate agreement, and values below 0.4 means poor agreement (White et al. 2013).
Analytical sensitivity. Clinical specimen matrices (self-collected vaginal swab, penile meatal swab, female urine, and male urine) collected in Aptima Multitest Swab and Aptima Urine Specimen transports (Hologic) were acquired through anonymous in-house collections from consenting volunteer donors and screened for the presence of M. genitalium using the AMG IVD assay. Negative specimens were then pooled and aliquoted before being used to prepare serial dilutions of M. genitalium strain M30 (ATCC catalog number 49895) at various concentrations. Aptima specimen transport medium (STM) (Hologic) was similarly used to prepare M. genitalium lysate dilutions as a “pure system” specimen type. M. genitalium strain M30 lysate preparations were quantitated for genomic DNA titer with an in-house developed real-time Invader chemistry (24) assay targeting the 23S rRNA gene, using uncut plasmid DNA calibration standards containing the M. genitalium 23S rRNA gene sequence, and quantitated on a NanoDrop instrument (Thermo Fisher). For all four TMA assays and all specimen matrix types, a minimum
Protein-protein interactions are important for most of the cellular processes in life. Hence, understanding the mechanism of protein-protein recognition at molecular level is of practical interest and has direct applications to functional genomics. Unravelling the mechanisms of protein-protein recognition is a fundamental problem, which would aid in function prediction and drug design. The availability of structures of numerous protein-pro- tein complexes in Protein Data Bank (PDB) enables researchers to analyze the binding sites in terms of
The introduction of peptide nucleicacid (PNA) has opened up exciting opportunities for DNA biosensors. The unique structural, hybridization, and recognition features of solution-phase PNA (12, 13) can be readily extrapolated onto transducer surfaces in connection with the design of DNA biosensors. Such use of surface-confined PNA recognition layers imparts remarkable sequence specificity onto DNA biosensors and offers other attractive advantages (including greater latitude in the selection of experimental conditions). The greatly improved distinction between closely related sequences accrued from the use of PNA were first realized in connection with electrochemical transduction of the PNA/DNA hybridization event (4), and subsequently using other types of biosensors (5, 6). This article reviews the status of PNA-based biosensors, and their prospects for future DNA diagnostics.
Catalysis. Catalysis is implemented by means of genelet activation, which requires completion of a single-stranded promoter region (Figure 10B). Two main challenges needed to be overcome in order to achieve signal-speci ﬁ c catalysis. First, it was necessary to ensure that the catalyst binds reversibly to the genelet, so that it is not consumed. Second, the sequence of the catalyst needed to be a subsequence of the promoter region. This presented a con ﬂ ict, since reversible binding required short sequences, while promoter completion required the presence of a speci ﬁ c promoter sequence. To reconcile these con ﬂ icting goals, we introduced a translator gate that took an RNA signal ⟨ xs x ⟩ as input and produced an intermediate DNA strand ⟨ t xs ⟩ as output, which contained the missing promoter sequence represented by the toehold t = TATTA. By using the same toehold t in all intermediate strands, a single promoter could be used for all of the genelets. The translator gate also ensured that the promoter was completed by a DNA strand rather than an RNA transcript, which circumvents potential issues related to additional bases that may result from nonspeci ﬁ c extension of RNA transcripts. 16 In order to ensure that only a fraction of the signal ⟨ xs x ⟩ was translated to intermediates, the toehold x 1 * on the translator gate was chosen to only complement a portion of the sequence x = x 1 x 2 , thus enabling toehold unbinding. Furthermore, an initial
Design of the oligochromatographic stick. The OC dipstick (Fig. 1) is double- sided, with a polymer (plastic) support backing (a). On either side of the support, several membranes and absorbents regulate the flow and allow sequential hy- bridization. (i) The lower absorbent pad (c) is impregnated with the detection probe coupled to gold particles (probe conjugate). The probe conjugate is dried in the lower absorbent; it will be solubilized when the OC dipstick is placed in the NASBA product mixed with running buffer. The control side contains the probe conjugate specific for the internal-control RNA, while the test side contains the probe conjugate specific for the target (T. brucei). (ii) The intermediate nitro- cellulose membrane (b) contains two capture zones on each side. The lower capture zones (d) allow the concentration of the nucleicacid detected. They are coated with the two specific capture probes binding the T. brucei amplicon on the test side (d1) and the internal-control RNA amplicon on the control side (d2). The upper capture zones, or migration control lines (e), allow the validation of the migration by capturing the excess probe conjugates on both sides. The capture elements on these zones are oligonucleotides complementary to the probe conjugates. (iii) The upper absorbents (f) allow the migration of liquid on the nitrocellulose membrane to continue by absorbing the excess.
Implications for Design of Nanodevices. For many design applications, nucleic acids rep- resent an attractive building material. Consider for example, an attempt to design a mechanical device that performs work by moving through a series of conformations. It would be cumbersome to parameterize the protein design problem for mechanical devices in terms of atomic coordinates. It also seems unlikely that it would be possible to conditionally stabilize a sequence of non-natural folds using positive design methods that do not explicitly treat fold specificity. However, DNA devices with moving parts and complex conditional conformational changes have already been de- signed (using ad hoc methods) and experimentally demonstrated [47, 43, 42]. We expect that nucleicacid secondary structure will provide a productive framework for formulating the design problem for functional multi-state machines in a way that simultaneously addresses positive and negative design requirements. Ultimately, the objective of rational nucleicaciddesign efforts is to develop a ‘molecular compiler’ that takes as input a conceptual design for a device and produces as output, a list of nucleicacid sequences that can be expected to assemble into the desired structures and function robustly.
Previously, we demonstrated the utility of a probe sequence reported in the patent literature, CalB2208, for the specific detec- tion of C. albicans via DNA-FISH (23). While we were able to increase the hybridization intensity using DNA helper probes, this probe was intrinsically dimmer than other probes evaluated in that study, including the positive control, EUK-516. We hypoth- esized that the use of PNA chemistry might improve the perfor- mance characteristics of a probe targeted to the CalB2208 region. An examination of the literature indicated that existing probes for C. albicans either targeted the small ribosomal subunit (18S) or FIG 3 Comparative staining of C. albicans-directed DNA- and PNA-FISH probes analyzed via flow cytometry. Probes D-CalB2208 and D-Ca720, associated DNA helpers, and probes P-CalB2208 and P-Ca726 were used. (A) Scatter plot data for treatments with the D-CalB2208 probe with C. dubliniensis ATCC MYA-180 (negative control) (a), the D-CalB2208 probe plus DNA helpers with C. albicans ATCC 90028 (b), and the P-CalB2208 probe with C. albicans ATCC 90028 (c). (B) Corresponding results for the D-Ca720 and P-Ca726 probes. Bar graphs to the right of each scatter plot provide quantitative comparisons of results for the hybridization of DNA probes, DNA probes plus helpers, or PNA probes against C. albicans ATCC 90028.
Besides simulated datasets, we tested our method using multiple sets of real biological sequences. One issue with real biological sequences is the lack of prior knowledge about the size and maximum numbers of mutations per- mitted by the motif. The optimal motif(s) comes from the model having the smallest neighborhood probability and produces the least number of motifs. In order to pin down the optimal motif, the algorithm must be run for a range of l and d. But we have found that the search of the opti- mal l and d can be done methodically by making use of the neighborhood probability of each model. In the situ- ation when iTriplet has found too many motifs for the specified model then we can conclude that the model is too lax and so a more stringent model should be used, by increasing l or reducing d or both at the same time. Alter- natively, once a satisfactory model is found, one can look for shorter models with similar neighborhood probability if the shorter alternative gives a similar result. In order to ease the effort for searching for the optimal model, iTri- plet provides an autonomous mode option. Under auton- omous mode, the program will explore various models using the strategy just described, and return the best mod- els with motif length from 6 to 40 bases and maximum number of differences from 1 to 12. But the user also has the option to limit the size of motif to a specific range. Although many models are examined, only a very limited numbers of models, usually none or one, can provide the optimal motif unless the given sequences contain multi- ple motifs. Several reasons are that a slight change in the size and/or the maximum number of mutations will result in a substantial change in neighborhood probability which can be seen in Table 2. As mentioned in the Back- ground section, we have included promoter and 5' UTR regions from four genes commonly chosen as test cases for motif finding algorithms [10,14,17]. In addition, we have also added a set of 3' UTR sequences in our test in order to understand how our method performs in other regions of a gene. Table 3 summarizes the prediction by iTriplet for various genes and genomic regions.
The isothermal nucleicacidsequence-based amplification (NASBA) system was applied for the detection of rhinoviruses using primers targeted at the 5 ⴕ noncoding region (5 ⴕ NCR) of the viral genome. The nucleotide sequence of the 5 ⴕ NCRs of 34 rhinovirus isolates was determined to map the most conserved regions and design more appropriate primers and probes. The assay amplified RNA extracted from 30 rhinovirus reference strains and 88 rhinovirus isolates, it did not amplify RNA from 49 enterovirus isolates and other respiratory viruses. The assay allows one to discriminate between group A and B rhinoviruses. Sensitivities for the detection of group B and group A rhinoviruses was 20 and 200 50% tissue culture infective doses, respectively.
inoculation, we reassessed the particle to infectivity (P/I) ratio and the maximum possible size of a prion-specific polynucle- otide. We found that polynucleotides larger than 25 bases were not in sufficient abundance to be present at a P/I ratio of 1 and thus could not be essential for prion infectivity. We also irra- diated two prion strains with UV light at 254 nm to determine if one strain was more susceptible to inactivation by UV radi- ation than the other. Our bioassay results indicate that the two strains were equally resistant to inactivation by UV radiation that was designed to target polynucleotides. Combined with recent studies on the production of synthetic prions that infect either mammals (29, 36, 37, 76) or fungi (34, 38, 70, 73), the results reported here argue persuasively that nucleic acids are not essential either for prion infectivity or for specifying strain- encrypted properties.
acids—D-glucuronic acid and D-galacturonic acid. D- glucuronic acid is common in hemicellulose polysaccha- rides, whereas D-galacturonic acid in the form of galac- turonans is the main component of pectic substances. It is worth noting that these polysaccharides can be easily obtained from oat seeds and apple pulp. Thus, acidic oligosaccharides were extracted from apples according to a standard method described earlier for oat seeds . The extracted PU was additionally treated with deoxyribo- nuclease and proteases to prevent contamination with endogenous DNA. The enzymes from PU samples were then eliminated by three-fold treatment with phenol- chloroform mixture followed by three-fold precipitation by triple volume cooled ethanol .
hand, in high-grade CIN and cancer, E6 and E7 are expressed throughout the thickness of the cervical epithelium. Therefore, E6/E7 mRNA has been proposed as a more specific marker for cervical dysplasia and cancer than HPV DNA (15). Multiplex nucleicacidsequence-based amplification (NASBA) assays, which utilize molecular beacon probes for the real-time detec- tion and typing of E6/E7 mRNA from HPV type 16 (HPV16), HPV18, HPV31, HPV33 and HPV45, are commercially avail- able (PreTect HPV-Proofer [NorChip AS, Klokkarstua, Nor- way] and NucliSENS EasyQ [bioMe ´rieux, Marcy l’Etoile, France]). In theory, the isothermal (41°C) NASBA technology only amplifies single stranded nucleic acids (NA) or RNA equivalents, even in a background of double-stranded DNA (dsDNA) (12). However, unexpected dsDNA amplification by NASBA has been reported, which demonstrates the necessity of verifying the origin of a NASBA signal when specific RNA detection is the objective (29).
Rapid and sensitive detection of Aspergillus from clinical samples may facilitate the early diagnosis of invasive pulmonary aspergillosis (IPA). A real-time nucleicacidsequence-based amplification (NASBA) method was investigated by use of an inhalational rat model of IPA. Immunosuppressed male Sprague-Dawley rats were exposed to Aspergillus fumigatus spores for an hour in an aerosol chamber. Bronchoalveolar lavage (BAL) fluid, lung tissues, and whole blood were collected from five infected rats at 1, 24, 48, 72, and 96 h postinfection and five uninfected rats at the end of the experiment. Total nucleicacid (TNA) was extracted on an easyMAG instrument. A primer-molecular beacon set targeting 28S rRNA was designed to detect Aspergillus spp. The results were compared to those of quantitative PCR (qPCR) (18S rDNA) and quantitative culture. The analytical sensitivity of the real-time NASBA assay was <1 CFU/assay. A linear range of detection was demonstrated over 5 log units of conidia (10 to 10 5 spores). Both NASBA and qPCR showed a progressive increase in lung tissue burdens, while the CFU counts were stable over time. The fungal burdens in BAL fluid were more variable and not indicative of a progressive infection. The results of both real-time assays correlated well for both sample types (r ⴝ 0.869 and P < 0.0001 for lung tissue, r ⴝ 0.887 and P < 0.0001 for BAL fluid). For all whole-blood specimens, NASBA identified Aspergillus-positive samples in the group from which samples were collected at 72 h postinfection (three of five samples) and the group from which samples were collected at 96 h postinfection (five of five samples), but no positive results were obtained by culture or PCR. Real-time NASBA is highly sensitive and useful for the detection of Aspergillus in an experimental model of IPA.
The sequencing chamber consists of a flexible plastic adhesive flow cell, less than a millimeter in height, which has been applied on the top of a low-autofluorescence microscope coverslip. The sequencing chamber holds approximately 80 µL of fluid. Template DNA is anchored to the surface via the strong binding properties of biotin and streptavidin. Biotin, a modified version containing an amine group, is first covalently bonded to the carboxyl groups of polyacrylic acid, the negatively charged polyelectrolyte, using the catalyst EDC. Next, streptavidin is bound to biotin on the coverslip surface. Template DNA, which has been biotinylated at its 5′ end, is finally applied onto the streptavidin. Thus, streptavidin acts as a bridge between the surface biotin and the biotin of the template DNA. The template DNA has previously been annealed to a Cy3-labeled primer.