Detection and quantification of Lyme spirochetes using sensitive and specific molecular beacon probes

<p>Abstract</p> <p>Background</p> <p>Lyme disease, caused by <it>Borrelia burgdorferi</it>, affects a large number of people in both the USA and Europe. The mouse is a natural host for this spirochete and is widely used as a model system to study Lyme pathogenesis mechanisms. Since disease manifestations often depend upon the spirochete burden in a particular tissue, it is critical to accurately measure the bacterial number in infected tissues. The current methods either lack sensitivity and specificity (SYBR Green), or require independent analysis of samples in parallel to quantitate host and bacterial DNA (TaqMan). We have developed a novel molecular beacon-based convenient multiplex real-time quantitative PCR assay to identify and detect small numbers of <it>B. burgdorferi </it>in infected mouse tissues.</p> <p>Results</p> <p>We show here that molecular beacons are effective, sensitive and specific probes for detecting and estimating wide-ranging numbers of <it>B. burgdorferi </it>in the presence of mouse DNA. In our assays, the spirochete <it>recA </it>and the mouse <it>nidogen </it>gene amplicons were detected simultaneously using molecular beacons labeled with different fluorophores. We further validated the application of these probes by quantifying the wild-type strain and <it>bgp</it>-defective mutant of <it>B. burgdorferi</it>. The <it>bgp</it>-defective mutant shows a ten-fold reduction in the level of spirochetes present in various tissues.</p> <p>Conclusion</p> <p>The high sensitivity and specificity of molecular beacons makes them superior probes for the detection of small numbers of <it>B. burgdorferi</it>. Furthermore, the use of molecular beacons can be expanded for the simultaneous detection and quantification of multiple pathogens in the infected hosts, including humans, and in the arthropod vectors.</p

Single-molecule imaging of transcriptionally coupled and uncoupled splicing.

Introns are removed from pre-mRNAs during transcription while the pre-mRNA is still tethered to the gene locus via RNA polymerase. However, during alternative splicing, it is important that splicing be deferred until all of the exons and introns involved in the choice have been synthesized. We have developed an in situ RNA imaging method with single-molecule sensitivity to define the intracellular sites of splicing. Using this approach, we found that the normally tight coupling between transcription and splicing is broken in situations where the intron's polypyrimidine tract is sequestered within strong secondary structures. We also found that in two cases of alternative splicing, in which certain exons are skipped due to the activity of the RNA-binding proteins Sxl and PTB, splicing is uncoupled from transcription. This uncoupling occurs only on the perturbed introns, whereas the preceding and succeeding introns are removed cotranscriptionally

An integrated biological approach to guide the development of metal-chelating inhibitors of influenza virus PA endonuclease

The influenza virus PA endonuclease, which cleaves capped cellular pre-mRNAs to prime viral mRNA synthesis, is a promising target for novel anti-influenza virus therapeutics. The catalytic center of this enzyme resides in the N-terminal part of PA (PA-Nter) and contains two (or possibly one or three) Mg(2+) or Mn(2+) ions which are critical for its catalytic function. There is large interest in PA inhibitors that are optimally designed to occupy the active site and chelate the metal ions. We here focused on a series of β-diketo acid (DKA) and DKA-bioisosteric compounds containing different scaffolds, and determined their structure-activity relationship in an enzymatic assay with PA-Nter, to build a 3D pharmacophore model. Besides, we developed a molecular beacon (MB)-based PA-Nter assay which enables to compare the inhibition versus Mn(2+) or Mg(2+), the latter probably being the biologically relevant cofactor. This real-time MB assay allows to measure the enzyme kinetics of PA-Nter or perform high-throughput screening. Several DKA derivatives were found to cause strong inhibition of PA-Nter with IC50 values comparable to that of the prototype L-742,001 (i.e. below 2 μM). Among the different compounds tested, L-742,001 appeared unique in having equal activity against either Mg(2+) or Mn(2+). Three compounds ( 10: , with a pyrrole scaffold, and 40: and 41: , with an indole scaffold) exhibited moderate antiviral activity in cell culture (EC99 values: 64-95 μM), and were proven to affect viral RNA synthesis. Our approach to integrate complementary enzymatic, cellular and mechanistic assays should guide ongoing development of improved influenza virus PA inhibitors.status: publishe