Axiomatically, the density of information stored in DNA, with just four nucleotides (GACT), is higher than in a binary code, but less than it might be if synthetic biologists succeed in adding independently replicating nucleotides to genetic systems. Such addition could also add additional functional groups, not found in natural DNA but useful for molecular performance. Here, we consider two new nucleotides (Z and P, 6-amino-5-nitro-3-(1′-β-D-2′-deoxyribo-furanosyl)-2(1H)-pyridone and 2-amino-8-(1′-β-D-2′-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one). These are designed to pair via strict Watson-Crick geometry. These were added to lies in a ibrarlaboratory in vitro evolution (LIVE) experiment; the GACTZP library was challenged to deliver molecules that bind selectively to liver cancer cells, but not to untransformed liver cells. Unlike in classical in vitro selection systems, low levels of mutation allow this system to evolve to create binding molecules not necessarily present in the original library. Over a dozen binding species were recovered. The best had Z and/or P in their sequences. Several had multiple, nearby, and adjacent Z’s and P’s. Only the weaker binders contained no Z or P at all. This suggests that this system explored much of the sequence space available to this genetic system, and that GACTZP libraries are richer reservoir of functionality than standard libraries.

The first general method for the selection of boronic acid-based aptamers that allow for glycan substructure focusing is described. Using fibrinogen as a model, we have selected boronic acid-modified DNA aptamers that have high affinities (low nM K d ) and the ability to recognize changes in the glycosylation site. The method developed should also be applicable to the development of aptamers for other glyco-products, such as glycolipids and glycopeptides. E-mail: wang@gsu.edu Supporting Information Available: Description of the experimental procedure and detailed results. This material is available free of charge via the Internet at http://pubs.acs.org. Glycosylation profoundly affects the function and activities of many proteins. 1,2 However, detecting and differentiating variations in glycosylation as an integral part of a glycoprotein is not a trivial matter, mostly due to a lack of good tools. Two most powerful methods exist for developing "binders" for glycoproteins: antibody production and nucleic acid-based aptamer selection. 2 However, none of these methods has the intrinsic ability to specifically focus on the glycosylation site, which include both the glycan and the surrounding structures, in epitope selection. We are interested in examining the possibility of directing the selection of aptamers to preferentially go after the glycosylation site of a glycoprotein (the sweet spot). By taking advantage of many published methods on incorporating modified nucleotide into DNA/RNA for aptamer selection, 3 we decided to incorporate a boronic acid-modified thymidine-5′-triphosphate (B-TTP, Figure 1) into DNA for aptamer selection. Because of the intrinsic ability for the boronic acid moiety to interact with diols 4 and single hydroxyl groups, 5 we hypothesized that the incorporation of the boronic acid moiety into DNA would allow the selection to gravitate toward the glycosylation site and therefore for the specific recognition of the glycosylation site. When necessary, counter selection can be used to eliminate unwanted cross-reactivity for binders as described in literatures. 3, 6 Herein, we report our work that demonstrates the feasibility by using a model protein, fibrinogen, which was chosen because of its commercial availability in large quantities and its known glycan structures. NIH Public AccessWe used the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) approach for aptamer selection. 2,3 Introduction of the boronic acid moiety was accomplished through tethering to the 5-position of TTP ( Figure 1) because (1) modification at this position has long been known to have minimal effect on polymerase-catalyzed incorporation; 3 (2) 5-position modified TTP has been widely used in aptamer selections to tune their affinity and bestow novel properties, 3 and (3) we have demonstrated that the B-TTP can be successfully incorporated into DNA using DNA polymerases, and the synthesized boronic acid-modified DNA (B-DNA) can serve as templates for further amplification. 7For the aptamer sel...

In addition to storage of genetic information, DNA can also catalyze various reactions. RNA-cleaving DNAzymes are the catalytic DNAs discovered the earliest, and they can cleave RNAs in a sequence-specific manner. Owing to their great potential in medical therapeutics, virus control, and gene silencing for disease treatments, RNA-cleaving DNAzymes have been extensively studied; however, the mechanistic understandings of their substrate recognition and catalysis remain elusive. Here, we report three catalytic form 8–17 DNAzyme crystal structures. 8–17 DNAzyme adopts a V-shape fold, and the Pb2+ cofactor is bound at the pre-organized pocket. The structures with Pb2+ and the modification at the cleavage site captured the pre-catalytic state of the RNA cleavage reaction, illustrating the unexpected Pb2+-accelerated catalysis, intrinsic tertiary interactions, and molecular kink at the active site. Our studies reveal that DNA is capable of forming a compacted structure and that the functionality-limited bio-polymer can have a novel solution for a functional need in catalysis.

Our understanding of membranes and membrane lipid function has lagged far behind that of nucleic acids and proteins, largely because it is difficult to manipulate cellular membrane lipid composition. To help solve this problem, we show that methyl-α-cyclodextrin (MαCD)-catalyzed lipid exchange can be used to maximally replace the sphingolipids and phospholipids in the outer leaflet of the plasma membrane of living mammalian cells with exogenous lipids, including unnatural lipids. In addition, lipid exchange experiments revealed that 70-80% of cell sphingomyelin resided in the plasma membrane outer leaflet; the asymmetry of metabolically active cells was similar to that previously defined for erythrocytes, as judged by outer leaflet lipid composition; and plasma membrane outer leaflet phosphatidylcholine had a significantly lower level of unsaturation than phosphatidylcholine in the remainder of the cell. The data also provided a rough estimate for the total cellular lipids residing in the plasma membrane (about half). In addition to such lipidomics applications, the exchange method should have wide potential for investigations of lipid function and modification of cellular behavior by modification of lipids.lipid exchange | plasma membrane outer leaflet | lipid asymmetry | methyl-alpha-cyclodextrin | mass spectrometry

The excessive activation of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors, a subtype of glutamate ion channels, has been implicated in various neurological diseases such as cerebral ischemeia and amyotrophic lateral sclerosis. Inhibitors of AMPA receptors are drug candidates for potential treatment of these diseases. Using the systematic evolution of ligands by exponential enrichment (SELEX), we have selected a group of RNA aptamers against the recombinant GluR2Qflip AMPA receptor transiently expressed in HEK-293 (human embryonic kidney) cells. One of the aptamers, AN58, is shown to competitively inhibit the receptor. The nanomolar affinity of AN58 rivals that of NBQX (6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione), one of the best competitive inhibitors. Like NBQX, AN58 has the highest affinity for GluR2, the selection target, among all AMPA receptor subunits. However, AN58 has a higher selectivity for the GluR4 AMPA receptor subunit and remains potent even at pH = 6.8 (i.e., a clinically relevant acidic pH), as compared with NBQX. Furthermore, this RNA molecule possesses stable physical properties. Therefore, AN58 serves as a unique lead compound for developing water-soluble inhibitors with a nanomolar affinity for GluR2 AMPA receptors.

Methyl-β-cyclodextrin (MβCD) can be used to exchange membrane lipids between different vesicles in order to prepare model membrane vesicles with lipid asymmetry. To help define what factors influence lipid exchange, we studied how lipid interaction with cyclodextrins (CDs) was affected by lipid and CD structure. The decrease in light scattering upon CD-induced vesicle solubilization and the change in Förster resonance energy transfer of labeled lipids upon vesicle solubilization and lipid exchange were used to detect phospholipid-CD interaction. Of the CDs examined, MβCD, hydroxypropyl-α-cyclodextrin (HPαCD), and hydroxypropyl-β-cyclodextrin (HPβCD) were the three with the most suitable phospholipid interaction properties. Only MβCD was observed to dissolve lipid vesicles (at least at CD concentrations below 125 mM). Solubilization of lipid vesicles was half complete at 10-80 mM MβCD with progressively higher MβCD concentrations required as phospholipid acyl chain length increased from 14 to 22 carbons. Phospholipid acyl chain unsaturation and lipid headgroup structure also affected the amount of MβCD needed for solubilization. All three CDs studied were able to carry out phospholipid exchange. MβCD, which retained the ability to carry out lipid exchange below MβCD concentrations needed for solubilization, exchanged lipid more efficiently than HPαCD or HPβCD. However, the ability of HPαCD to exchange phospholipids, coupled with its inability to interact with cholesterol, indicates that it will be useful for preparing asymmetric vesicles with controlled amounts of cholesterol.

Ample evidence from earlier studies of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, GluR3 included, suggests that alternative splicing not only enriches AMPA receptor diversity but also, more importantly, creates receptor variants that are functionally different. However, it is not known whether alternative splicing affects the receptor channel opening that occurs in the microsecond time domain. Using a laser-pulse photolysis technique combined with whole-cell recording, we characterized the channel opening rate process for two alternatively spliced variants of GluR3, i.e., GluR3flip and GluR3flop. We show that the alternative splicing that generates flip and flop variants of GluR3 receptors regulates the channel opening process by controlling the rate of channel closing but not the rate of channel opening or the glutamate binding affinity. Specifically, the flop variant closes its channel almost 4-fold faster than the flip variant. We therefore propose that the function of the flip-flop sequence module in the channel opening process of AMPA receptors is to stabilize the open channel conformation, presumably by its pivotal structural location. Furthermore, a comparison of the flip isoform among all AMPA receptor subunits, based on the magnitude of the channel opening rate constant, suggests that GluR3 is kinetically more similar to GluR2 and GluR4 than to GluR1.

The T−A and C−G base pairing and stacking allow the formation of the stable DNA duplex structure for genetic information storage, transcription, and replication. To replace the oxygen of the nucleotide nucleobases with selenium for the studies of the base-pair recognition, the duplex stability, and the nuclei acid crystal structures, we have synthesized for the first time the 4-Se thymidine phosphoramidite and incorporated it into oligonucleotides via solid-phase synthesis with high coupling yield (99%). The Se modification on the nucleobase is relatively stable under the elevated temperature. Using the dUSe (2‘-Se-dU) to facilitate the crystallization, we have successfully crystallized the DNA containing the 4-Se−T substitution and determined its structure at 1.50 Å resolution. The UV-melting and X-ray crystal structure studies have indicated that the Se substitution on the nucleobase does not cause a significant structure perturbation, the large Se atom on the thymine can be successfully accommodated by the DNA duplex, and the Se-mediated hydrogen bond (longer than the usual hydrogen bond) is formed within the modified T−A base pair. In addition, the Se derivatization on the nucleobases further facilitates X-ray crystal structure determination of nucleic acids and their protein complexes via Se MAD phasing.