Transformation of personal computers and mobile phones into genetic diagnostic systems.

Molecular diagnostics based on the polymerase chain reaction (PCR) offer rapid and sensitive means for detecting infectious disease, but prohibitive costs have impeded their use in resource-limited settings where such diseases are endemic. In this work, we report an innovative method for transforming a desktop computer and a mobile camera phone--devices that have become readily accessible in developing countries--into a highly sensitive DNA detection system. This transformation was achieved by converting a desktop computer into a de facto thermal cycler with software that controls the temperature of the central processing unit (CPU), allowing for highly efficient PCR. Next, we reconfigured the mobile phone into a fluorescence imager by adding a low-cost filter, which enabled us to quantitatively measure the resulting PCR amplicons. Our system is highly sensitive, achieving quantitative detection of as little as 9.6 attograms of target DNA, and we show that its performance is comparable to advanced laboratory instruments at approximately 1/500th of the cost. Finally, in order to demonstrate clinical utility, we have used our platform for the successful detection of genomic DNA from the parasite that causes Chagas disease, Trypanosoma cruzi, directly in whole, unprocessed human blood at concentrations 4-fold below the clinical titer of the parasite.

Development of an efficient targeted cell-SELEX procedure for DNA aptamer reagents.

BACKGROUND: DNA aptamers generated by cell-SELEX offer an attractive alternative to antibodies, but generating aptamers to specific, known membrane protein targets has proven challenging, and has severely limited the use of aptamers as affinity reagents for cell identification and purification. METHODOLOGY: We modified the BJAB lymphoblastoma cell line to over-express the murine c-kit cell surface receptor. After six rounds of cell-SELEX, high-throughput sequencing and bioinformatics analysis, we identified aptamers that bound BJAB cells expressing c-kit but not wild-type BJAB controls. One of these aptamers also recognizes c-kit endogenously expressed by a mast cell line or hematopoietic progenitor cells, and specifically blocks binding of the c-kit ligand stem cell factor (SCF). This aptamer enables better separation by fluorescence-activated cell sorting (FACS) of c-kit(+) hematopoietic progenitor cells from mixed bone marrow populations than a commercially available antibody, suggesting that this approach may be broadly useful for rapid isolation of affinity reagents suitable for purification of other specific cell types. CONCLUSIONS/SIGNIFICANCE: Here we describe a novel procedure for the efficient generation of DNA aptamers that bind to specific cell membrane proteins and can be used as high affinity reagents. We have named the procedure STACS (Specific TArget Cell-SELEX).

Selection is more intelligent than design: improving the affinity of a bivalent ligand through directed evolution.

Multivalent molecular interactions can be exploited to dramatically enhance the performance of an affinity reagent. The enhancement in affinity and specificity achieved with a multivalent construct depends critically on the effectiveness of the scaffold that joins the ligands, as this determines their positions and orientations with respect to the target molecule. Currently, no generalizable design rules exist for construction of an optimal multivalent ligand for targets with known structures, and the design challenge remains an insurmountable obstacle for the large number of proteins whose structures are not known. As an alternative to such design-based strategies, we report here a directed evolution-based method for generating optimal bivalent aptamers. To demonstrate this approach, we fused two thrombin aptamers with a randomized DNA sequence and used a microfluidic in vitro selection strategy to isolate scaffolds with exceptionally high affinities. Within five rounds of selection, we generated a bivalent aptamer that binds thrombin with an apparent dissociation constant (K(d)) <10 pM, representing a ∼200-fold improvement in binding affinity over the monomeric aptamers and a ∼15-fold improvement over the best designed bivalent construct. The process described here can be used to produce high-affinity multivalent aptamers and could potentially be adapted to other classes of biomolecules.

Selection strategy to generate aptamer pairs that bind to distinct sites on protein targets.

Many analytical techniques benefit greatly from the use of affinity reagent pairs, wherein each reagent recognizes a discrete binding site on a target. For example, antibody pairs have been widely used to dramatically increase the specificity of enzyme linked immunosorbent assays (ELISA). Nucleic acid-based aptamers offer many advantageous features relative to protein-based affinity reagents, including well-established chemical synthesis, thermostability, and low production cost. However, the generation of suitable aptamer pairs has posed a significant challenge, and few such pairs have been reported to date. To address this important challenge, we present multivalent aptamer isolation systematic evolution of ligands by exponential enrichment (MAI-SELEX), a technique designed for the efficient selection of aptamer pairs. In contrast to conventional selection methods, our method utilizes two selection modules to generate separate aptamer pools that recognize distinct binding sites on a single target. Using MAI-SELEX, we have isolated two groups of 2'-fluoro-modified RNA aptamers that specifically recognize the αV or ß3 subunits of integrin αVß3. These aptamers exhibit low nanomolar affinities for their targets, with minimal cross-reactivity to other closely related integrin homologues. Moreover, we show that these aptamer pairs do not interfere with each other's binding and effectively detect the target even in complex mixtures such as undiluted serum.

Probing the limits of aptamer affinity with a microfluidic SELEX platform.

Nucleic acid-based aptamers offer many potential advantages relative to antibodies and other protein-based affinity reagents, including facile chemical synthesis, reversible folding, improved thermal stability and lower cost. However, their selection requires significant time and resources and selections often fail to yield molecules with affinities sufficient for molecular diagnostics or therapeutics. Toward a selection technique that can efficiently and reproducibly generate high performance aptamers, we have developed a microfluidic selection process (M-SELEX) that can be used to obtain high affinity aptamers against diverse protein targets. Here, we isolated DNA aptamers against three protein targets with different isoelectric points (pI) using a common protocol. After only three rounds of selection, we discovered novel aptamer sequences that bind to platelet derived growth factor B (PDGF-BB; pI = 9.3) and thrombin (pI = 8.3) with respective dissociation constants (K(d)) of 0.028 nM and 0.33 nM, which are both superior to previously reported aptamers against these targets. In parallel, we discovered a new aptamer that binds to apolipoprotein E3 (ApoE; pI = 5.3) with a K(d) of 3.1 nM. Furthermore, we observe that the net protein charge may exert influence on the affinity of the selected aptamers. To further explore this relationship, we performed selections against PDGF-BB under different pH conditions using the same selection protocol, and report an inverse correlation between protein charge and aptamer K(d).

Improving aptamer selection efficiency through volume dilution, magnetic concentration, and continuous washing in microfluidic channels.

The generation of nucleic acid aptamers with high affinity typically entails a time-consuming, iterative process of binding, separation, and amplification. It would therefore be beneficial to develop an efficient selection strategy that can generate these high-quality aptamers rapidly, economically, and reproducibly. Toward this goal, we have developed a method that efficiently generates DNA aptamers with slow off-rates. This methodology, called VDC-MSELEX, pairs the volume dilution challenge process with microfluidic separation for magnetic bead-assisted aptamer selection. This method offers improved aptamer selection efficiencies through the application of highly stringent selection conditions: it retrieves a small number (<10(6)) of magnetic beads suspended in a large volume (>50 mL) and concentrates them into a microfluidic chamber (8 µL) with minimal loss for continuous washing. We performed three rounds of the VDC-MSELEX using streptavidin (SA) as the target and obtained new DNA aptamer sequences with low nanomolar affinity that specifically bind to the SA proteins.

Efficiency of synaptic transmission of single-photon events from rod photoreceptor to rod bipolar dendrite.

A rod transmits absorption of a single photon by what appears to be a small reduction in the small number of quanta of neurotransmitter (Q(count)) that it releases within the integration period ( approximately 0.1 s) of a rod bipolar dendrite. Due to the quantal and stochastic nature of release, discrete distributions of Q(count) for darkness versus one isomerization of rhodopsin (R*) overlap. We suggested that release must be regular to narrow these distributions, reduce overlap, reduce the rate of false positives, and increase transmission efficiency (the fraction of R* events that are identified as light). Unsurprisingly, higher quantal release rates (Q(rates)) yield higher efficiencies. Focusing here on the effect of small changes in Q(rate), we find that a slightly higher Q(rate) yields greatly reduced efficiency, due to a necessarily fixed quantal-count threshold. To stabilize efficiency in the face of drift in Q(rate), the dendrite needs to regulate the biochemical realization of its quantal-count threshold with respect to its Q(count). These considerations reveal the mathematical role of calcium-based negative feedback and suggest a helpful role for spontaneous R*. In addition, to stabilize efficiency in the face of drift in degree of regularity, efficiency should be approximately 50%, similar to measurements.

A clockwork hypothesis: synaptic release by rod photoreceptors must be regular.

We can see at light intensities much lower than an average of one photon per rod photoreceptor, demonstrating that rods must be able to transmit a signal after absorption of a single photon. However, activation of one rhodopsin molecule (Rh*) hyperpolarizes a mammalian rod by just 1 mV. Based on the properties of the voltage-dependent Ca2+ channel and data on [Ca2+] in the rod synaptic terminal, the 1 mV hyperpolarization should reduce the rate of release of quanta of neurotransmitter by only 20%. If quantal release were Poisson, the distributions of quantal count in the dark and in response to one Rh* would overlap greatly. Depending on the threshold quantal count, the overlap would generate too frequent false positives in the dark, too few true positives in response to one Rh*, or both. Therefore, quantal release must be regular, giving narrower distributions of quantal count that overlap less. We model regular release as an Erlang process, essentially a mechanism that counts many Poisson events before release of a quantum of neurotransmitter. The combination of appropriately narrow distributions of quantal count and a suitable threshold can give few false positives and appropriate (e.g., 35%) efficiency for one Rh*.

Cell density ratios in a foveal patch in macaque retina.

We examine the assumptions that the fovea contains equal numbers of inner (invaginating or ON) and outer (flat or OFF) midget bipolar cells and equal numbers of inner and outer diffuse bipolar cells. Based on reconstruction from electron photomicrographs of serial thin sections through the fovea of a macaque monkey, we reject both assumptions. First, every foveal L and M cone is presynaptic to one inner and one outer midget bipolar cell; however, S cones are presynaptic to one outer but no inner midget bipolar cell. Second, we measure the density of all foveal cells in the same patch of fovea, affording accurate cell density ratios. For each foveal cone pedicle, at a density of 26,500 mm(-2), there is close to one (0.88) outer diffuse bipolar cell but only 0.40 inner diffuse bipolar cells. This asymmetry may be related to differences in resolution and sensitivity for light increments and decrements. We also find one (1.01) Müller cell, one (1.01) amacrine cell in the inner nuclear layer, and close to one (0.83) horizontal cell for each cone pedicle. In addition, for each S cone, there are two inner S-cone bipolar cells and two small bistratified ganglion cells. In total, there are 3.4 cone bipolar cells per cone but only 2.6 ganglion cells per cone. The latter ratio is enough to accommodate one midget ganglion cell for each midget bipolar cell.