Inosine is an important RNA modification, furthermore RNA oxidation has gained interest due, in part, to its potential role in the development/progression of disease as well as on its impact on RNA structure and function. In this report we established the base pairing abilities of purine nucleobases G, I, A, as well as their corresponding, 8-oxo-7,8-dihydropurine (common products of oxidation at the C8-position of purines), and 8-bromopurine (as probes to explore conformational changes), derivatives, namely 8-oxoG, 8-oxoI, 8-oxoA, 8-BrG, and 8-BrI. Dodecamers of RNA were obtained using standard phosphoramidite chemistry via solid-phase synthesis, and used as models to establish the impact that each of these nucleobases have on the thermal stability of duplexes, when base pairing to canonical and noncanonical nucleobases. Thermal stabilities were obtained from thermal denaturation transition (T m) measurements, via circular dichroism (CD). The results were then rationalized using models of base pairs between two monomers, via density functional theory (DFT), that allowed us to better understand potential contributions from H-bonding patterns arising from distinct conformations. Overall, some of the important results indicate that: (a) an anti-I:syn-A base pair provides thermal stability, due to the absence of the exocyclic amine; (b) 8-oxoG base pairs like U, and does not induce destabilization within the duplex when compared to the pyrimidine ring; (c) a U:G wobble-pair is only stabilized by G; and (d) 8-oxoA displays an inherited base pairing promiscuity in this sequence context. Gaining a better understanding of how this oxidatively generated lesions potentially base pair with other nucleobases will be useful to predict various biological outcomes, as well as in the design of biomaterials and/or nucleotide derivatives with biological potential.
Inosine is ubiquitous and essential in many biological processes, including RNA-editing. In addition, oxidative stress on RNA has been a topic of increasing interest due, in part, to its potential role in the development/progression of disease. In this work we probed the ability of three reverse transcriptases (RTs) to catalyze the synthesis of cDNA in the presence of RNA templates containing inosine (I), 8-oxo-7,8-dihydroinosine (8oxo-I), guanosine (G), or 8-oxo-7,8-dihydroguanosine (8-oxoG), and explored the impact that these purine derivatives have as a function of position. To this end, we used 29-mers of RNA (as template) containing the modifications at position-18 and reverse transcribed DNA using 17-mers, 18mers, or 19-mers (as primers). Generally reactivity of the viral RTs, AMV / HIV / MMLV, towards cDNA synthesis was similar for templates containing G or I as well as for those with 8-oxoG or 8-oxoI. Notable differences are: 1) the use of 18-mers of DNA (to explore cDNA synthesis past the lesion/modification) led to inhibition of DNA elongation in cases where a G:dA wobble pair was present, while the presence of I, 8-oxoI, or 8-oxoG led to full synthesis of the corresponding cDNA, with the latter two displaying a more efficient process; 2) HIV RT is more sensitive to modified base pairs in the vicinity of cDNA synthesis; and 3) the presence of a modification two positions away from transcription initiation has an adverse impact on the overall process. Steady-state kinetics were established using AMV RT to determine substrate specificities towards canonical dNTPs (N = G, C, T, A). Overall we found evidence that RNA templates containing inosine are likely to incorporate dC > dT > > dA, where reactivity in the presence of dA was found to be pH dependent (process abolished at pH 7.3); and that the absence of the C2-exocyclic amine, as displayed with templates containing 8-oxoI, leads to increased selectivity towards incorporation of dA over dC. The data will be useful in assessing the impact that the presence of inosine and/or oxidatively generated lesions have on viral processes and adds to previous reports where I codes exclusively like G. Similar results were obtained upon comparison of AMV and MMLV RTs.
The complete structural analysis and resynthesis of an unknown compound, selected from a library of dipeptides, provided a framework for a course that built from expository experiments toward a guided-inquiry approach. We used this semester-long lab as a replacement for conventional second semester organic chemistry laboratory and successfully covered the critical concepts and lab techniques typically covered in traditional courses while better engaging the students.
PurposeThis study aims to compare radiographic outcomes and complication rates of immobilization with an abduction pillow to spica casting for postoperative care after a hip reconstruction with varus derotational proximal femur osteotomy (VDRO) with or without pelvic osteotomy for children with cerebral palsy (CP).Methods233 children (1–18 years old) diagnosed with CP that underwent VDRO with or without pelvic osteotomy were identified, of which 188 patients were immobilized with a spica cast and 45 were immobilized with an abduction pillow, based on surgeon preference. 123 (65%) in the Spica group and 21 (47%) in the pillow group had pelvic osteotomies. Demographic data and complication rates were collected. Radiographic parameters, including anatomic medial proximal femoral angle (aMPFA), acetabular index (AI) and migration percentage (MP), were measured for each patient at the completion of surgery, six weeks post-operatively, and one year post-operatively.ResultsThere was not a statistically significant difference in BMI (p = 0.285), gender distribution (p = 0.984), or median follow-up time (p = 0.314) between groups. Rates of complications were consistent among groups with no differences in instances of delayed unions (p = 0.10), subluxations (p = 0.55), infection (p = 0.71), or non-unions (p = 0.10). There was no statistically significant difference in number of patients with an ideal aMPFA, AI, or MP (p = 0.44, p = 0.19, p = 1.00) at one year post-operatively.ConclusionsImmobilization with an abduction pillow is a safe and effective alternative to hip spica casting following hip reconstruction.
Scheme1.Oxidation of Ga tthe C8-positionleads to 8-oxoG. The groupa tthisposition experiences steric hindrance with the C5'-hydrogen atoms, and this induces ac onformational changet ot he syn isomer. Scheme2.A)Sequence of theophylline aptamer and structures of xanthine derivatives. B) Intrastrand/intermolecular interactions (color-coded)that are directly involved in theophylline recognition.Scheme3.Sequences of hairpins 5-8 mimicking the upper scaffold ofthe aptamerand their corresponding T m values (experimentally measured and calculated with UNAFOLD). Conditions: RNA 3.5 mm,NaCl 1mm,MgCl 2 5mm,Na 2 P 2 O 7 10 mm,p H7.2.Scheme4.Representations of RNA aptamer modified at position A) G25 (2/8), or B) G26 (3/9)a nd proposed models of how 8-oxoG might induce aunique tertiarystructure thatdisrupts the binding pocket. C) Plausible structures after modificationa tG11,s howing how 8-oxoG is likely to have ad ifferent, more pronounced, impacto nt he overall structure. Small moleculeConc. range theophylline 11.25 mm-343 nm theobromine 1mm-30 nm caffeine 11.25 mm-343 nm
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