Gel purification of radiolabeled nucleic acids via phosphorimaging: Dip-N-Dot.

RNA and DNA oligonucleotides radiolabeled with (32)P or (33)P often require gel electrophoresis to remove undesired side and/or degradation products. Common ways to visualize these molecules after electrophoresis are by ultraviolet (UV) shadowing, which necessarily reduces the specific activity of the oligonucleotide, and by autoradiography using film, which is cumbersome and increases the cost of generating the radiolabeled molecule. A more cost-effective method is to physically inject the gel with a "Dip-N-Dot" solution of dye and radionuclide after electrophoresis but prior to phosphorimaging. The gel can be overlaid on its computer-generated image, allowing the labeled molecules to be visualized quickly.

Daily functioning and quality of life in children with sickle cell disease pain: relationship with family and neighborhood socioeconomic distress.

UNLABELLED: The aim of this study was to examine the relationship between individual/family and neighborhood socioeconomic distress, pain, and functional outcomes in children with sickle cell disease (SCD). We hypothesized that both individual economic distress as well as residence in neighborhoods of severe economic distress would predict children's level of pain-related functional disability and health-related quality of life (HRQOL). Participants (mean age, 12.14 years; 57% male, n = 56) were recruited from an outpatient hematology clinic at a Midwestern tertiary referral hospital. Questionnaires assessing pain, depression, functional disability, and HRQOL were completed by children and their caregivers. Individual socioeconomic data including parental education and family income were reported by caregivers. Neighborhood socioeconomic distress was identified using publicly available census tract data and was based on neighborhood poverty, female head of household, male unemployment, and high school dropout levels. Multivariate regression analyses revealed that individual/family socioeconomic distress was a significant predictor of children's functional disability and physical and psychosocial HRQOL. Neighborhood socioeconomic distress emerged as a significant independent predictor of physical HRQOL only, where living in a distressed neighborhood predicted diminished physical HRQOL. Findings suggest that individual socioeconomic status and neighborhood economic distress play similar but independent roles in predicting children's functional outcomes related to SCD pain. PERSPECTIVE: Little is known about the influence of either individual/family or neighborhood socioeconomic factors on pain and functioning in children with SCD. Our findings suggest that socioeconomic distress defined at both the individual level and at the neighborhood/community level are significant independent predictors of pain-related disability and HRQOL in children with SCD.

RNA-directed construction of structurally complex and active ligase ribozymes through recombination.

RNA-directed recombination can be used to catalyze a disproportionation reaction among small RNA substrates to create new combinations of sequences. But the accommodation of secondary and tertiary structural constraints in the substrates by recombinase ribozymes has not been explored. Here, we show that the Azoarcus group I intron can recombine oligoribonucleotides to construct class I ligase ribozymes, which are catalytically active upon synthesis. The substrate oligonucleotides, ranging in size from 58 to 104 nucleotides (nt), along with the 152-nt ligase ribozymes they reconstitute, can contain significant amounts of secondary structure. However, substrate recognition by the Azoarcus ribozyme depends on the existence of a single accessible CAU triplet for effective recombination. A biphasic temperature reaction profile was designed such that the sequential recombination/ligation events could take place in a thermocycler without human intervention. A temperature-dependent pH shift of the reaction buffer contributes to the success of the net reaction. When the substrate for the ligase ribozyme is introduced into the reaction mixture, as much as 11% can be observed being converted to product by the recombined ligase in the same reaction vessel. Recombination followed by ligation can also occur under isothermal conditions at 37 degrees C. Tertiary structure formation of the ligase upon construction can provide some protection from cleavage by the Azoarcus ribozyme when compared to the constituent substrates. These data suggest that RNA-directed recombination can, in fact, articulate complex ribozymes, and that there are logical rules that can guide the optimal placement of the CAU recognition sequence.

Expanded divalent metal-ion tolerance of evolved ligase ribozymes.

Class I ligases are artificial ribozymes that catalyze the joining of two single-stranded RNAs. These ribozymes are between 120 and 160 nucleotides in length, making them intermediate in size for catalytic RNAs. Previous characterization of the b1-207 ribozyme suggests that it behaves similar to larger ribozymes in terms of divalent metal-ion dependence. This molecule displays a strong preference for magnesium for catalysis, and is inactive in any other metal except manganese, which actually inhibits its operation in magnesium. Here, we sought to examine the metal-ion usages of two ligases that were obtained through continuous evolution in vitro from the b1-207 sequence framework. We found an expanded catalytic range for the E(100)(#3) and B(16)(#19) ribozymes, as they are both catalytically active in calcium and strontium, and less inhibited by manganese. Though not selected for activity in these salts, the evolved ribozymes exhibit several adaptations to in vitro catalysis, and their ability to accommodate metals other than magnesium can be viewed as an example of a molecular exaptation.

Generalized RNA-directed recombination of RNA.

RNA strand exchange through phosphor-nucleotidyl transfer reactions is an intrinsic chemistry promoted by group I intron ribozymes. We show here that Tetrahymena and Azoarcus ribozymes can promote RNA oligonucleotide recombination in either two-pot or one-pot schemes. These ribozymes bind one oligonucleotide, cleave following a guide sequence, transfer the 3' portion of the oligo to their own 3' end, bind a second oligo, and catalyze another transfer reaction to generate recombinant oligos. Recombination is most effective with the Azoarcus ribozyme in a single reaction vessel in which over 75% of the second oligo can be rapidly converted to recombinant product. The Azoarcus ribozyme can also create a new functional RNA, a hammerhead ribozyme, which can be constructed via recombination and then immediately promote its own catalysis in a homogeneous milieu, mimicking events in a prebiotic soup.