Penultimate Base Research Articles

ALLELE-SPECIFIC QPCR METHOD FOR SNP AND INDEL GENOTYPING BASED ON FRET

The proposed Allele-specific q-PCR (ASQ) method for genotyping of single nucleotide polymorphism (SNP) and insertion-deletion polymorphism (InDel), based on Fluorescence resonance energy transfer (FRET). Initially named by its developers as Kompetitive Allele Specific PCR (KASP), this method is an AS-PCR variant adapted for fluorescence-based detection of amplification results. We developed a bioinformatic tool for designing probe sequences for PCR-based genotyping assays. Probe sequences are designed in both directions, and both single nucleotide polymorphisms (SNPs) and insertion-deletions (InDels) may be targeted. In addition, the tool allows discrimination of up to four allelic variants at a single SNP site. To increase both the reaction specificity and the discriminative power of SNP genotyping, each allele-specific primer is designed such that the penultimate base before the primer’s 3′ end base is positioned at the SNP site. The tool allows the design of custom FRET cassette reporter systems for fluorescence-based assays. FastPCR is a user-friendly and powerful Java-based software that is freely available (http://primerdigital.com/tools/). Using the FastPCR environment and the tool for designing AS-PCR provides unparalleled flexibility for developing genotyping assays and specific and sensitive diagnostic PCR-based tests, translating into a greater likelihood of research success.

Nearest neighbor rules for RNA helix folding thermodynamics: improved end effects.

Nearest neighbor parameters for estimating the folding stability of RNA secondary structures are in widespread use. For helices, current parameters penalize terminal AU base pairs relative to terminal GC base pairs. We curated an expanded database of helix stabilities determined by optical melting experiments. Analysis of the updated database shows that terminal penalties depend on the sequence identity of the adjacent penultimate base pair. New nearest neighbor parameters that include this additional sequence dependence accurately predict the measured values of 271 helices in an updated database with a correlation coefficient of 0.982. This refined understanding of helix ends facilitates fitting terms for base pair stacks with GU pairs. Prior parameter sets treated 5′GGUC3′ paired to 3′CUGG5′ separately from other 5′GU3′/3′UG5′ stacks. The improved understanding of helix end stability, however, makes the separate treatment unnecessary. Introduction of the additional terms was tested with three optical melting experiments. The average absolute difference between measured and predicted free energy changes at 37°C for these three duplexes containing terminal adjacent AU and GU pairs improved from 1.38 to 0.27 kcal/mol. This confirms the need for the additional sequence dependence in the model.

Designing Allele-Specific Competitive-Extension PCR-Based Assays for High-Throughput Genotyping and Gene Characterization.

Polymerase chain reaction (PCR) is a simple and rapid method that can detect nucleotide polymorphisms and sequence variation in basic research applications, agriculture, and medicine. Variants of PCR, collectively known as allele-specific PCR (AS-PCR), use a competitive reaction in the presence of allele-specific primers to preferentially amplify only certain alleles. This method, originally named by its developers as Kompetitive Allele Specific PCR (KASP), is an AS-PCR variant adapted for fluorescence-based detection of amplification results. We developed a bioinformatic tool for designing probe sequences for PCR-based genotyping assays. Probe sequences are designed in both directions, and both single nucleotide polymorphisms (SNPs) and insertion-deletions (InDels) may be targeted. In addition, the tool allows discrimination of up to four-allelic variants at a single SNP site. To increase both the reaction specificity and the discriminative power of SNP genotyping, each allele-specific primer is designed such that the penultimate base before the primer’s 3′ end base is positioned at the SNP site. The tool allows design of custom FRET cassette reporter systems for fluorescence-based assays. FastPCR is a user-friendly and powerful Java-based software that is freely available (http://primerdigital.com/tools/). Using the FastPCR environment and the tool for designing AS-PCR provides unparalleled flexibility for developing genotyping assays and specific and sensitive diagnostic PCR-based tests, which translates into a greater likelihood of research success.

Modified “Allele-Specific qPCR” Method for SNP Genotyping Based on FRET

The proposed method is a modified and improved version of the existing “Allele-specific q-PCR” (ASQ) method for genotyping of single nucleotide polymorphism (SNP) based on fluorescence resonance energy transfer (FRET). This method is similar to frequently used techniques like Amplifluor and Kompetitive allele specific PCR (KASP), as well as others employing common universal probes (UPs) for SNP analyses. In the proposed ASQ method, the fluorophores and quencher are located in separate complementary oligonucleotides. The ASQ method is based on the simultaneous presence in PCR of the following two components: an allele-specific mixture (allele-specific and common primers) and a template-independent detector mixture that contains two or more (up to four) universal probes (UP-1 to 4) and a single universal quencher oligonucleotide (Uni-Q). The SNP site is positioned preferably at a penultimate base in each allele-specific primer, which increases the reaction specificity and allele discrimination. The proposed ASQ method is advanced in providing a very clear and effective measurement of the fluorescence emitted, with very low signal background-noise, and simple procedures convenient for customized modifications and adjustments. Importantly, this ASQ method is estimated as two- to ten-fold cheaper than Amplifluor and KASP, and much cheaper than all those methods that rely on dual-labeled probes without universal components, like TaqMan and Molecular Beacons. Results for SNP genotyping in the barley genes HvSAP16 and HvSAP8, in which stress-associated proteins are controlled, are presented as proven and validated examples. This method is suitable for bi-allelic uniplex reactions but it can potentially be used for 3- or 4-allelic variants or different SNPs in a multiplex format in a range of applications including medical, forensic, or others involving SNP genotyping.

Parallel loss of introns in the ABCB1 gene in angiosperms

BackgroundThe presence of non-coding introns is a characteristic feature of most eukaryotic genes. While the size of the introns, number of introns per gene and the number of intron-containing genes can vary greatly between sequenced eukaryotic genomes, the structure of a gene with reference to intron presence and positions is typically conserved in closely related species. Unexpectedly, the ABCB1 (ATP-Binding Cassette Subfamily B Member 1) gene which encodes a P-glycoprotein and underlies dwarfing traits in maize (br2), sorghum (dw3) and pearl millet (d2) displayed considerable variation in intron composition.ResultsAn analysis of the ABCB1 gene structure in 80 angiosperms revealed that the number of introns ranged from one to nine. All introns in ABCB1 underwent either a one-time loss (single loss in one lineage/species) or multiple independent losses (parallel loss in two or more lineages/species) with the majority of losses occurring within the grass family. In contrast, the structure of the closest homolog to ABCB1, ABCB19, remained constant in the majority of angiosperms analyzed. Using known phylogenetic relationships within the grasses, we determined the ancestral branch-points where the losses occurred. Intron 7, the longest intron, was lost in only a single species, Mimulus guttatus, following duplication of ABCB1. Semiquantitative PCR showed that the M. guttatus ABCB1 gene copy without intron 7 had significantly lower transcript levels than the gene copy with intron 7. We further demonstrated that intron 7 carried two motifs that were highly conserved across the monocot-dicot divide.ConclusionsThe ABCB1 gene structure is highly dynamic, while the structure of ABCB19 remained largely conserved through evolution. Precise removal of introns, preferential removal of smaller introns and presence of at least 2 bp of microhomology flanking most introns indicated that intron loss may have predominantly occurred through non-homologous end-joining (NHEJ) repair of double strand breaks. Lack of microhomology in the exon upstream of lost phase I introns was likely due to release of the selective constraint on the penultimate base (3rd base in codon) of the terminal codon by the splicing machinery. In addition to size, the presence of regulatory motifs will make introns recalcitrant to loss.

Nonhomologous end joining of complex DNA double-strand breaks with proximal thymine glycol and interplay with base excision repair.

DNA double-strand breaks induced by ionizing radiation are often accompanied by ancillary oxidative base damage that may prevent or delay their repair. In order to better define the features that make some DSBs repair-resistant, XLF-dependent nonhomologous end joining of blunt-ended DSB substrates having the oxidatively modified nonplanar base thymine glycol at the first (Tg1), second (Tg2), third (Tg3) or fifth (Tg5) positions from one 3' terminus, was examined in human whole-cell extracts. Tg at the third position had little effect on end-joining even when present on both ends of the break. However, Tg as the terminal or penultimate base was a major barrier to end joining (>10-fold reduction in ligated products) and an absolute barrier when present at both ends. Dideoxy trapping of base excision repair intermediates indicated that Tg was excised from Tg1, Tg2 and Tg3 largely if not exclusively after DSB ligation. However, Tg was rapidly excised from the Tg5 substrate, resulting in a reduced level of DSB ligation, as well as slow concomitant resection of the opposite strand. Ligase reactions containing only purified Ku, XRCC4, ligase IV and XLF showed that ligation of Tg3 and Tg5 was efficient and only partially XLF-dependent, whereas ligation of Tg1 and Tg2 was inefficient and only detectable in the presence of XLF. Overall, the results suggest that promoting ligation of DSBs with proximal base damage may be an important function of XLF, but that Tg can still be a major impediment to repair, being relatively resistant to both trimming and ligation. Moreover, it appears that base excision repair of Tg can sometimes interfere with repair of DSBs that would otherwise be readily rejoined.

Structures and Energetics of Four Adjacent G·U Pairs That Stabilize an RNA Helix.

Consecutive G·U base pairs inside RNA helices can be destabilizing, while those at the ends of helices are thermodynamically stabilizing. To determine if this paradox could be explained by differences in base stacking, we determined the high-resolution (1.32 Å) crystal structure of (5'-GGUGGCUGUU-3')2 and studied three sequences with four consecutive terminal G·U pairs by NMR spectroscopy. In the crystal structure of (5'-GGUGGCUGUU-3')2, the helix is overwound but retains the overall features of A-form RNA. The penultimate base steps at each end of the helix have high base overlap and contribute to the unexpectedly favorable energetic contribution for the 5'-GU-3'/3'-UG-5' motif in this helix position. The balance of base stacking and helical twist contributes to the positional dependence of G·U pair stabilities. The energetic stabilities and similarity to A-form RNA helices suggest that consecutive G·U pairs would be recognized by RNA helix binding proteins, such as Dicer and Ago. Thus, these results will aid future searches for target sites of small RNAs in gene regulation.

Abstract 3028: Tolerance and intolerance of proximal thymine glycol in DNA double-strand break repair by nonhomologous end joining

Abstract Double-strand breaks (DSBs) induced by ionizing radiation are often accompanied by ancillary oxidative base damage that could prevent or delay their repair. In order to better define the features that make some DSBs repair-resistant, plasmid DSB substrates were constructed with two blunt ends, one having the oxidatively modified nonplanar base thymine glycol at the first, second or third positions from the 3′ terminus (Tg1, Tg2 and Tg3, respectively). End joining was examined in extracts of Bustel fibroblasts, wherein end joining is completely dependent on addition of exogenous recombinant XLF, and therefore reflects classical nonhomologous end joining. Tg at the third position had almost no effect on end-joining even when present on both ends of the break. However, the Tg1 and Tg2 substrates yielded tenfold to twentyfold less head-to-tail joins and no detectable head-to-head joins, suggesting that Tg as the terminal or penultimate base is a serious barrier to NHEJ and an absolute barrier when present at both ends. For all substrates, Tg-containing ligation products predominated at early times, and remarkably, a 3′ terminal Tg was in some cases directly ligated to an undamaged blunt end. Dideoxy trapping of base excision repair intermediates indicated that Tg was excised in the extracts, but largely if not exclusively after DSB ligation. For all substrates, accurate blunt end ligation without end trimming predominated. However addition of excess Artemis nuclease to the extracts resulted in a slightly shorter ligation product, and a slight increase in yield, for the Tg2 substrate only. For the Tg1 substrate, there was a distinct 2-hr delay between addition of substrate and the detection of ligated products, while the unmodified and Tg3 products showed a much shorter delay of ∼30 min. Overall, the results suggest that Tg can be a major impediment to DSB repair when present as the terminal or penultimate base, being relatively resistant to both trimming and ligation. Citation Format: Duaa Bafail, Sri Lakshmi Chalasani, Mohammed Al mohaini, Konstantin Akopiants, Lawrence F. Povirk. Tolerance and intolerance of proximal thymine glycol in DNA double-strand break repair by nonhomologous end joining. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3028. doi:10.1158/1538-7445.AM2015-3028

Characterization of a novel CAPN3 transcript generated by alternative splicing in cattle.

Calpain-3 (CAPN3) is a member of the calpain family of Ca(2+)-regulated cysteine proteases, which play an important role in sarcomere remodeling and mitochondrial protein turnover, and thus, regulating beef tenderness in cattle. Currently, multiple CAPN3 transcripts have been detected in human, monkey, rat, and rabbit. However, whether this transcript is present in cattle remains unknown. In this study, we identified 2 CAPN3 transcripts in the skeletal muscle individuals of local black cattle from Jilin, China. One transcript corresponded to the known full-length protein and was referred to as CAPN3a, while the second transcript did not contain exons 2-19 and contained a single-nucleotide insert in the penultimate base of exon 1 compared to CAPN3a; this protein was referred to as CAPN3b. The expression level of CAPN3b was approximately 50-fold lower than that of CAPN3a. Moreover, CAPN3b mRNA was not translated into a functional protein because it had lost essential domains according to bioinformatic analysis. Our results not provide a foundation for understanding the function of CAPN3, but also are useful for further elucidating the effect of CAPN3 on meat quality in cattle.

RB69 DNA Polymerase Structure, Kinetics, and Fidelity

This review will summarize our structural and kinetic studies of RB69 DNA polymerase (RB69pol) as well as selected variants of the wild-type enzyme that were undertaken to obtain a deeper understanding of the exquisitely high fidelity of B family replicative DNA polymerases. We discuss how the structures of the various RB69pol ternary complexes can be used to rationalize the results obtained from pre-steady-state kinetic assays. Our main findings can be summarized as follows. (i) Interbase hydrogen bond interactions can increase catalytic efficiency by 5000-fold; meanwhile, base selectivity is not solely determined by the number of hydrogen bonds between the incoming dNTP and the templating base. (ii) Minor-groove hydrogen bond interactions at positions n – 1 and n – 2 of the primer strand and position n – 1 of the template strand in RB69pol ternary complexes are essential for efficient primer extension and base selectivity. (iii) Partial charge interactions among the incoming dNTP, the penultimate base pair, and the hydration shell surrounding the incoming dNTP modulate nucleotide insertion efficiency and base selectivity. (iv) Steric clashes between mismatched incoming dNTPs and templating bases with amino acid side chains in the nascent base pair binding pocket (NBP) as well as weak interactions and large gaps between the incoming dNTPs and the templating base are some of the reasons that incorrect dNTPs are incorporated so inefficiently by wild-type RB69pol. In addition, we developed a tC°–tCnitro Förster resonance energy transfer assay to monitor partitioning of the primer terminus between the polymerase and exonuclease subdomains.

DNA Methylation Detection and Inhibitor Screening Based on the Discrimination of the Aggregation of Long and Short DNA on a Negatively Charged Indium Tin Oxide Microelectrode

On the basis of the different diffusivity and existence of electrostatic repulsion between long and short DNA on the negatively changed indium tin oxide (ITO) microelectrode, a simple but sensitive immobilization free solution-phase electrochemical method for DNA methylation detection and inhibitor screening has been developed. Electroactive substance (methylene blue) tagged at the penultimate base T close to the 3'-terminal first, in the absence of DNA methylation, methylene blue-labeled electroactive fragments cannot be generated by Dpn I and results in a weak electrochemical response being detected on the ITO electrode. On the contrary, a remarkable electrochemical response can be achieved by the cleavage in the presence of DNA methylation since methylene blue-labeled electroactive fragments can be generated and aggregate on the ITO electrode. The proposed system does not need complex operation procedures such as bisulfite treatment, PCR amplification, and electrode immobilization. Six ITO microelectrodes had been assembled on the same microchip, which can achieve the parallel detection of the same sample and improve the experimental efficiency of drug screening. The system was used to conveniently and specifically monitor the change of the DNA methylation level with high sensitivity and selectivity. The proposed system has the potential application to screen the drugs as inhibitors on the activity of methyltransferase in the clinic.

Base Pair Opening in a Deoxynucleotide Duplex Containing a cis-syn Thymine Cyclobutane Dimer Lesion

The cis-syn thymine cyclobutane dimer is a DNA photoproduct implicated in skin cancer. We compared the stability of individual base pairs in thymine dimer-containing duplexes to undamaged parent 10-mer duplexes. UV melting thermodynamic measurements, CD spectroscopy, and 2D NOESY NMR spectroscopy confirm that the thymine dimer lesion is locally and moderately destabilizing within an overall B-form duplex conformation. We measured the rates of exchange of individual imino protons by NMR using magnetization transfer from water and determined the equilibrium constant for the opening of each base pair K(op). In the normal duplex K(op) decreases from the frayed ends of the duplex toward the center, such that the central TA pair is the most stable with a K(op) of 8 × 10⁻⁷. In contrast, base pair opening at the 5'T of the thymine dimer is facile. The 5'T of the dimer has the largest equilibrium constant (K(op) = 3 × 10⁻⁴) in its duplex, considerably larger than even the frayed penultimate base pairs. Notably, base pairing by the 3'T of the dimer is much more stable than by the 5'T, indicating that the predominant opening mechanism for the thymine dimer lesion is not likely to be flipping out into solution as a single unit. The dimer asymmetrically affects the stability of the duplex in its vicinity, destabilizing base pairing on its 5' side more than on the 3' side. The striking differences in base pair opening between parent and dimer duplexes occur independently of the duplex-single strand melting transitions.

Contribution of Partial Charge Interactions and Base Stacking to the Efficiency of Primer Extension at and beyond Abasic Sites in DNA

During DNA synthesis, base stacking and Watson-Crick (WC) hydrogen bonding increase the stability of nascent base pairs when they are in a ternary complex. To evaluate the contribution of base stacking to the incorporation efficiency of dNTPs when a DNA polymerase encounters an abasic site, we varied the penultimate base pairs (PBs) adjacent to the abasic site using all 16 possible combinations. We then determined pre-steady-state kinetic parameters with an RB69 DNA polymerase variant and solved nine structures of the corresponding ternary complexes. The efficiency of incorporation for incoming dNTPs opposite an abasic site varied between 2- and 210-fold depending on the identity of the PB. We propose that the A rule can be extended to encompass the fact that DNA polymerase can bypass dA/abasic sites more efficiently than other dN/abasic sites. Crystal structures of the ternary complexes show that the surface of the incoming base was stacked against the PB's interface and that the kinetic parameters for dNMP incorporation were consistent with specific features of base stacking, such as surface area and partial charge-charge interactions between the incoming base and the PB. Without a templating nucleotide residue, an incoming dNTP has no base with which it can hydrogen bond and cannot be desolvated, so that these surrounding water molecules become ordered and remain on the PB's surface in the ternary complex. When these water molecules are on top of a hydrophobic patch on the PB, they destabilize the ternary complex, and the incorporation efficiency of incoming dNTPs is reduced.

Real-Time PCR-Based Mismatch Amplification Mutation Assay for Specific Detection of CS6-Expressing Allelic Variants of Enterotoxigenic Escherichia coli and Its Application in Assessing Diarrheal Cases and Asymptomatic Controls

Enterotoxigenic Escherichia coli (ETEC) expressing the colonization factor CS6 is widespread in many developing countries, including India. The different allelic variants of CS6, caused by point mutations in its structural genes, cssA and cssB, are designated AIBI, AIIBII, AIIIBI, AIBII, and AIIIBII. A simple, reliable, and specific mismatch amplification mutation assay based on real-time quantitative PCR (MAMA-qPCR) was developed for the first time for the detection of CS6-expressing ETEC, along with the identification of allelic variations. The assay was based on mismatched nucleotide incorporation at the penultimate base at the 3' ends of the reverse primers specific for cssA and cssB and was validated using 38 CS6-expressing ETEC isolates. This strategy was effective in detecting all the alleles containing single-nucleotide polymorphisms. Using MAMA-qPCR, we also tested CS6 allelic variants in 145 ETEC isolates from children with acute diarrhea and asymptomatic infections, with the latter serving as controls. We observed that the AIBI and AIIIBI allelic variants were mostly associated with cases rather than controls, whereas the AIIBII variants were detected mostly in controls. In addition, the AIBI and AIIIBI alleles were frequently associated with ETEC harboring the heat-stable toxin gene (est) alone or with the heat-labile toxin gene (elt), whereas the AIIBII allele was predominant in ETEC isolates harboring the elt gene. This study may help in understanding the association of allelic variants in CS6-expressing ETEC with the clinical features of diarrhea, as well as in ETEC vaccine studies.

Substitution by Inosine at the 3′-Ultimate and Penultimate Positions of 16S rRNA Gene Universal Primers

Universal 16S rRNA gene primers (8F and 518R) bearing inosine substitutions at either the 3'-ultimate or the 3'-ultimate and penultimate base positions were exploited for the first time to study the bacterial community associated with coral polymicrobial Black Band Disease (BBD). Inosine-modified universal primer pairs display some shifting in the composition of 16S rRNA gene libraries, as well as expanding the observed diversity of a BBD bacterial community at the family/class level. Possible explanations for the observed shifts are discussed. These results thus point to the need for adopting multiple approaches in designing 16S rRNA universal primers for PCR amplification and subsequent construction of 16S rRNA gene libraries or pyrosequencing in the exploration of complex microbial communities.

The Effect of Primer-Template Mismatches on the Detection and Quantification of Nucleic Acids Using the 5′ Nuclease Assay

Real-time polymerase chain reaction (PCR) is the current method of choice for detection and quantification of nucleic acids, especially for molecular diagnostics. Complementarity between primers and template is often crucial for PCR applications, as mismatches can severely reduce priming efficiency. However, little quantitative data on the effect of these mismatches is available. We quantitatively investigated the effects of primer-template mismatches within the 3'-end primer region on real-time PCR using the 5'-nuclease assay. Our results show that single mismatches instigate a broad variety of effects, ranging from minor (<1.5 cycle threshold, eg, A-C, C-A, T-G, G-T) to severe impact (>7.0 cycle threshold, eg, A-A, G-A, A-G, C-C) on PCR amplification. A clear relationship between specific mismatch types, position, and impact was found, which remained consistent for DNA versus RNA amplifications and Taq/Moloney murine leukemia virus versus rTth based amplifications. The overall size of the impact among the various master mixes used differed substantially (up to sevenfold), and for certain master mixes a reverse or forward primer-specific impact was observed, emphasizing the importance of the experimental conditions used. Taken together these data suggest that mismatch impact follows a consistent pattern and enabled us to formulate several guidelines for predicting primer-template mismatch behavior when using specific 5-nuclease assay master mixes. Our study provides novel insight into mismatch behavior and should allow for more optimized development of real-time PCR assays involving primer-template mismatches.

A NewFgf10Mutation in the Mouse Leads to Atrophy of the Harderian Gland and Slit-Eye Phenotype in Heterozygotes: A Novel Model for Dry-Eye Disease?

The purpose of the present study was to characterize a new slit-eye phenotype in the mouse. Genomewide linkage analysis was performed, and a candidate gene was sequenced. Eyes of the mutants were described morphologically, histologically, and by in situ hybridization. To allow morphologic and functional studies of the retina, mutants were outcrossed to C57BL/6. Within an ongoing ethyl-nitrosourea mutagenesis screen with C3HeB/FeJ mice, the authors identified a new mutant (referred to as Aey17) showing a slit-eye phenotype in heterozygotes; homozygous mutants are not viable because of major developmental defects. This mutation was mapped to the distal end of mouse chromosome 13, suggesting Fgf10 (encoding the fibroblast growth factor 10) as a candidate gene. An A-->G transition in the penultimate base of the first intron of Fgf10 leading to aberrant splicing with an additional 49 bp in exon 2 and to a frameshift with a premature stop codon after 54 new amino acids was identified. Histologic analysis of the major ocular tissues (cornea, lens, retina) did not reveal major alterations compared with the wild type, but the size of the Harderian gland was remarkably reduced in heterozygotes. Although Fgf10 was expressed in the developing retina, neither electroretinography nor the virtual drum indicated any abnormalities in heterozygous mutants; overall eye size was identical in wild types and heterozygotes. The mutation in the Fgf10 gene leads to a dominant slit-eye phenotype caused by atrophy of the Harderian gland.

Aminoacyl-tRNA recognition by the FemXWv transferase for bacterial cell wall synthesis

Transferases of the Fem family catalyse peptide-bond formation by using aminoacyl-tRNAs and peptidoglycan precursors as donor and acceptor substrates, respectively. The specificity of Fem transferases is essential since mis-incorporated amino acids could act as chain terminators thereby preventing formation of a functional stress-bearing peptidoglycan network. Here we have developed chemical acylation of RNA helices with natural and non-proteinogenic amino acids to gain insight into the specificity of the model transferase FemXWv. Combining modifications in the RNA and aminoacyl moieties of the donor substrate revealed that unfavourable interactions of FemXWv with the acceptor arm of tRNAGly and with l-Ser or larger residues quantitatively accounts for the preferential transfer of l-Ala observed with complete aminoacyl-tRNAs. The main FemXWv identity determinant was identified as the penultimate base pair (G2-C71) of the acceptor arm instead of G3•U70 for the alanyl-tRNA synthetase. FemXWv tolerated a configuration inversion of the Cα of l-Ala but not the introduction of a second methyl on this atom. These results indicate that aminoacyl-tRNA recognition by FemXWv is distinct from other components of the translation machinery and relies on the exclusion of bulky amino acids and of the sequence of tRNAGly from the active site.

WASP: a Web-based Allele-Specific PCR assay designing tool for detecting SNPs and mutations.

BackgroundAllele-specific (AS) Polymerase Chain Reaction is a convenient and inexpensive method for genotyping Single Nucleotide Polymorphisms (SNPs) and mutations. It is applied in many recent studies including population genetics, molecular genetics and pharmacogenomics. Using known AS primer design tools to create primers leads to cumbersome process to inexperience users since information about SNP/mutation must be acquired from public databases prior to the design. Furthermore, most of these tools do not offer the mismatch enhancement to designed primers. The available web applications do not provide user-friendly graphical input interface and intuitive visualization of their primer results.ResultsThis work presents a web-based AS primer design application called WASP. This tool can efficiently design AS primers for human SNPs as well as mutations. To assist scientists with collecting necessary information about target polymorphisms, this tool provides a local SNP database containing over 10 million SNPs of various populations from public domain databases, namely NCBI dbSNP, HapMap and JSNP respectively. This database is tightly integrated with the tool so that users can perform the design for existing SNPs without going off the site. To guarantee specificity of AS primers, the proposed system incorporates a primer specificity enhancement technique widely used in experiment protocol. In particular, WASP makes use of different destabilizing effects by introducing one deliberate 'mismatch' at the penultimate (second to last of the 3'-end) base of AS primers to improve the resulting AS primers. Furthermore, WASP offers graphical user interface through scalable vector graphic (SVG) draw that allow users to select SNPs and graphically visualize designed primers and their conditions.ConclusionWASP offers a tool for designing AS primers for both SNPs and mutations. By integrating the database for known SNPs (using gene ID or rs number), this tool facilitates the awkward process of getting flanking sequences and other related information from public SNP databases. It takes into account the underlying destabilizing effect to ensure the effectiveness of designed primers. With user-friendly SVG interface, WASP intuitively presents resulting designed primers, which assist users to export or to make further adjustment to the design. This software can be freely accessed at .

Folding Cooperativity in RNA and DNA Is Dependent on Position in the Helix

Secondary structural motifs play essential roles in the folding and function of RNA and DNA molecules. Previous work from our lab compared the folding of small DNA and RNA hairpin loops containing a sheared GA pair [Moody, E. M., Feerar, J. C., and Bevilacqua, P. C. (2004) Biochemistry 43, 7992-7998]. We found that the small DNA hairpins fold in a highly cooperative manner with indirect coupling, while their RNA counterparts fold in a much less cooperative fashion and display direct coupling. Herein, we extend this study to the double-stranded helix. We carried out double mutant cycles on base pairs having identical nearest-neighbor contexts but located in either external or internal helical registers. In the external register, both RNA and DNA exhibit extensive folding cooperativity between the penultimate and terminal base pair, which is independent of mismatch identity. In contrast, DNA exhibits virtually no folding cooperativity in the center of the helix, while RNA maintains substantial coupling, which is dependent on mismatch identity. Two models account for these non-nearest-neighbor effects: one involves the unfavorable entropy of helix initiation common to DNA and RNA, and the other involves steric and electrostatic strain peculiar to RNA. These data show that RNA can display cooperativity less than, greater than, or equal to that of DNA depending on context and position.