Development of a quantitative PCR assay for residual mouse DNA and comparison of four sample purification methods for DNA isolation.

Reliable and sensitive assays are required to determine whether a pharmaceutical product meets current regulatory guidelines for residual host cell DNA. In this study, the sensitivity of the qPCR assay was significantly improved by targeting the repetitive elements of mouse genome. This improved method allowed for sensitive and accurate quantitation of mouse genomic DNA in the range of 1 to 10(6)pg/mL. In addition, four sample purification methods for DNA isolation (Wako DNA extractor kit, MasterPure™ DNA purification kit, PrepSEQ™ residual DNA sample preparation kit, and phenol-chloroform extraction method with addition of glycogen), each representing a different strategy for DNA isolation from proteinaceous solutions, were evaluated by isolating DNA from a mouse monoclonal IgG antibody. Among these methods, Wako DNA extractor kit and MasterPure™ DNA purification kit demonstrated superior DNA recovery, repeatability, and sensitivity, with quantitation limits of 1pg/mL. To further evaluate these two DNA isolation methods, six replicates of an unspiked mouse polyclonal IgG antibody sample were tested by both methods, and both methods demonstrated a good degree of precision. Therefore, the residual mouse DNA quantitation methods described here represented rapid, accurate, precise, and sensitive procedures that can be used in quality control testing for regulatory compliance in the pharmaceutical industry.

Multivector fluorescence analysis of the xpt guanine riboswitch aptamer domain and the conformational role of guanine.

Purine riboswitches are RNA regulatory elements that control purine metabolism in response to intracellular concentrations of the purine ligands. Conformational changes of the guanine riboswitch aptamer domain induced by guanine binding lead to transcriptional regulation of genes involved in guanine biosynthesis. The guanine riboswitch aptamer domain has three RNA helices designated P1, P2, and P3. An overall model for the Mg(2+)- and guanine-dependent relative orientations and dynamics of P1, P2, and P3 has not been reported, and the conformational role of guanine under physiologically relevant conditions has not been fully elucidated. In this study, an ensemble and single-molecule fluorescence resonance energy transfer (FRET) study was performed on three orthogonally labeled variants of the xpt guanine riboswitch aptamer domain. The combined FRET data support a model in which the unfolded state of the aptamer domain has a highly dynamic P2 helix that switches rapidly between two orientations relative to nondynamic P1 and P3. At <<1 mM Mg(2+) (in the presence of a saturating level of guanine) or >or=1 mM Mg(2+) (in the absence of guanine), the riboswitch starts to adopt a folded conformation in which loop-loop interactions lock P2 and P3 into place. At >5 mM Mg(2+), further compaction occurs in which P1 more closely approaches P3. Our data help to explain the biological role of guanine as stabilizing the globally folded aptamer domain conformation at physiologically relevant Mg(2+) concentrations (

Transposition of two amino acids changes a promiscuous RNA binding protein into a sequence-specific RNA binding protein.

In yeast (Saccharomyces cerevisiae), the branchpoint binding protein (BBP) recognizes the conserved yeast branchpoint sequence (UACUAAC) with a high level of specificity and affinity, while the human branchpoint binding protein (SF1) binds the less-conserved consensus branchpoint sequence (CURAY) in human introns with a lower level of specificity and affinity. To determine which amino acids in BBP provide the additional specificity and affinity absent in SF1, a panel of chimeric SF1 proteins was tested in RNA binding assays with wild-type and mutant RNA substrates. This approach revealed that the QUA2 domain of BBP is responsible for the enhanced RNA binding affinity and specificity displayed by BBP compared with SF1. Within the QUA2 domain, a transposition of adjacent arginine and lysine residues is primarily responsible for the switch in RNA binding between BBP and SF1. Alignment of multiple branchpoint binding proteins and the related STAR/GSG proteins suggests that the identity of these two amino acids and the RNA target sequences of all of these proteins are correlated.