Recent Efforts and Milestones for Simulating Nucleic Acid FRET Experiments through Computational Methods.

Computational methods can greatly aid nucleic acid fluorescence experiments by either offering fully detailed atomic insights into the conformations and interactions present in the studied system or by providing accurate simulations of the fundamental parameters. Fluorescence-based optical biosensors show great potential for clinical diagnosis of life-altering diseases with a very high specificity. Many of the designs for such rely on the concept of Förster resonance energy transfer (FRET). Currently, the methods used experimentally make use of theoretical assumptions which fundamentally affect the results. Having a detailed atomistic overview or significant simulated parameters could improve the understanding of the calculations and provide much more accurate outcomes. However, there are many challenges that need to be addressed before standardized computational protocols can be employed. This review is meant to highlight the progress made for computational methods used to simulate FRET experiments for nucleic acid probes. Recent advances have been made in computational tools, such as force field parametrizations and improved protocols. Complementary simulations to experimental data are also comprised in the this review.

Label-Free Homogeneous microRNA Detection in Cell Culture Medium Based on Graphene Oxide and Specific Fluorescence Quenching.

Label-free homogeneous optical detection of low concentration of oligonucleotides using graphene oxide in complex solutions containing proteins remains difficult. We used a colloidal graphene oxide (GO) as a fluorescent probe quencher to detect microRNA-21 spiked-in cell culture medium, overcoming previously reported problematic aspects of protein interference with graphene oxide. We used a "turn off" assay for specific quenching-based detection of oligo DNA-microRNA hybridization in solution. A fluorescein conjugated 30-mer single-stranded DNA (ssDNA) probe was combined with a complementary synthetic microRNA (18 nucleotides) target. The probe-target hybridization was detected by specific quenching due to photoinduced electron transfer (PET). On the next step, GO captures and quenches the unhybridized probe by fluorescence resonance energy transfer (FRET) in the presence of cell culture medium supplemented with platelet lysate, 0.1% sodium dodecyl sulfate (SDS), 0.1% Triton X-100 and 50% formamide. This resulted in sensitive measurement of the specific probe-target complexes remaining in solution. The detection is linear in the range of 1 nM and 8 nM in a single 100 µL total volume assay sample containing 25% cell culture medium supplemented with platelet lysate. We highlight a general approach that may be adopted for microRNA target detection within complex physiological media.