Probing the RNA binding surface of the HIV-1 nucleocapsid protein by site-directed mutagenesis.

The highly conserved nucleocapsid protein domain in HIV-1 recognizes and binds SL3 in genomic RNA. In this work, we used the structure of the NCp7-SL3 RNA complex to guide the construction of 16 NCp7 mutants to probe the RNA binding surface of the protein [De Guzman, R. N., et al. (1998) Science 279, 384-388]. Thirteen residues with functional or structural significance were mutated individually to Ala (Asn(5), Phe(6), Val(13), Phe(16), Asn(17), Gly(19), Glu(21), Ile(24), Gln(45), Met(46), Gly(22), Pro(31), and Gly(40)), and three salt bridge switch mutants exchanged Lys and Glu (Lys(14)-Glu(21), Lys(33)-Glu(42), and Lys(38)-Glu(51)). Dissociation constants (Kd) determined by fluorescence titration and isothermal titration calorimetry were used to compare affinities of SL3 for the variant proteins to that for the wild type. The F16A (Phe(16) to Ala) variant showed a 25-fold reduction in affinity, consistent with a loss of organized structure in f1, the protein's first zinc finger. I24A, Q45A, and M46A reduced affinity by 2-5-fold; these residues occupy nearly equivalent positions in f1 and f2. E21A increased affinity by 3-fold, perhaps because of the mutant's increased net positive charge. Among the salt bridge switch mutants, only K14E/E21K in f1 caused a substantial change in affinity (5-fold reduction), binding SL3 with a biphasic binding isotherm. Aside from these six variants, most of the mutations studied have relatively minor effects on the stability of the complex. We conclude that many side chain interactions in the wild-type complex contribute little to stability or can be compensated by new contacts in the mutants.

Sampling a biomarker of the human immunodeficiency virus across a synthetic nanopore.

One primary goal in nanobiotechnology is designing new methodologies for molecular biomedical diagnosis at stages much earlier than currently possible and without use of expensive reagents and sophisticated equipment. In this work, we show the proof of principle for single-molecule detection of the nucleocapsid protein 7 (NCp7), a protein biomarker of the HIV-1 virus, using synthetic nanopores and the resistive-pulse technique. The biosensing mechanism relied upon specific interactions between NCp7 and aptamers of stem-loop 3 (SL3) in the packaging domain of the retroviral RNA genome. One critical step of this study was the choice of the optimal size of the nanopores for accurate, label-free determinations of the dissociation constant of the NCp7 protein-SL3 RNA aptamer complex. Therefore, we systematically investigated the NCp7 protein-SL3 RNA aptamer complex employing two categories of nanopores in a silicon nitride membrane: (i) small, whose internal diameter was smaller than 6 nm, and (ii) large, whose internal diameter was in the range of 7 to 15 nm. Here, we demonstrate that only the use of nanopores with an internal diameter that is smaller than or comparable with the largest cross-sectional size of the NCp7-SL3 aptamer complex enables accurate measurement of the dissociation constant between the two interacting partners. Notably, this determination can be accomplished without the need for prior nanopore functionalization. Moreover, using small solid-state nanopores, we demonstrate the ability to detect drug candidates that inhibit the binding interactions between NCp7 and SL3 RNA by using a test case of N-ethylmaleimide.

Engineering a rigid protein tunnel for biomolecular detection.

One intimidating challenge in protein nanopore-based technologies is designing robust protein scaffolds that remain functionally intact under a broad spectrum of detection conditions. Here, we show that an extensively engineered bacterial ferric hydroxamate uptake component A (FhuA), a ß-barrel membrane protein, functions as a robust protein tunnel for the sampling of biomolecular events. The key implementation in this work was the coupling of direct genetic engineering with a refolding approach to produce an unusually stable protein nanopore. More importantly, this nanostructure maintained its stability under many experimental circumstances, some of which, including low ion concentration and highly acidic aqueous phase, are normally employed to gate, destabilize, or unfold ß-barrel membrane proteins. To demonstrate these advantageous traits, we show that the engineered FhuA-based protein nanopore functioned as a sensing element for examining the proteolytic activity of an enzyme at highly acidic pH and for determining the kinetics of protein-DNA aptamer interactions at physiological salt concentration.

Acyclic identification of aptamers for human alpha-thrombin using over-represented libraries and deep sequencing.

BACKGROUND: Aptamers are oligonucleotides that bind proteins and other targets with high affinity and selectivity. Twenty years ago elements of natural selection were adapted to in vitro selection in order to distinguish aptamers among randomized sequence libraries. The primary bottleneck in traditional aptamer discovery is multiple cycles of in vitro evolution. METHODOLOGY/PRINCIPAL FINDINGS: We show that over-representation of sequences in aptamer libraries and deep sequencing enables acyclic identification of aptamers. We demonstrated this by isolating a known family of aptamers for human α-thrombin. Aptamers were found within a library containing an average of 56,000 copies of each possible randomized 15mer segment. The high affinity sequences were counted many times above the background in 2-6 million reads. Clustering analysis of sequences with more than 10 counts distinguished two sequence motifs with candidates at high abundance. Motif I contained the previously observed consensus 15mer, Thb1 (46,000 counts), and related variants with mostly G/T substitutions; secondary analysis showed that affinity for thrombin correlated with abundance (K(d) = 12 nM for Thb1). The signal-to-noise ratio for this experiment was roughly 10,000∶1 for Thb1. Motif II was unrelated to Thb1 with the leading candidate (29,000 counts) being a novel aptamer against hexose sugars in the storage and elution buffers for Concanavilin A (K(d) = 0.5 µM for α-methyl-mannoside); ConA was used to immobilize α-thrombin. CONCLUSIONS/SIGNIFICANCE: Over-representation together with deep sequencing can dramatically shorten the discovery process, distinguish aptamers having a wide range of affinity for the target, allow an exhaustive search of the sequence space within a simplified library, reduce the quantity of the target required, eliminate cycling artifacts, and should allow multiplexing of sequencing experiments and targets.

Effects of the nature and concentration of salt on the interaction of the HIV-1 nucleocapsid protein with SL3 RNA.

The mature nucleocapsid protein of HIV-1, NCp7, and the NC domains in gag precursors are attractive targets for anti-AIDS drug discovery. The stability of the 1:1 complex of NCp7 with a 20mer mimic of stem-loop 3 RNA (SL3, also called psi-RNA, in the packaging domain of genomic RNA) is strongly affected by changes in ionic strength. NC domains recognize and specifically package genomic HIV-1 RNA, while electrostatic attractions and high concentrations of protein and RNA drive NCp7 to completely coat the RNA in the mature virion. The specific interactions of NCp7 binding to loop bases of SL3 produce 1:1 complexes in solutions that have a NaCl concentration of >or=0.2 M, while the electrostatic interactions can dominate at

A nucleic acid switch triggered by the HIV-1 nucleocapsid protein.

A unimolecular oligonucleotide switch, termed here an AlloSwitch, binds the mature HIV-1 nucleocapsid protein, NCp7. This switch can be used as an indicator for the presence of free NCp7 and NC domains in precursor and fusion proteins. It is thermodynamically stable in two conformations, H and O. A FRET pair is covalently attached to the strands to report on the molecular state of the switch. The results show that NC has an affinity for O 170 times higher than its affinity for H and that in the absence of NC the equilibrium ratio K1 = [O]/[H] = 0.10 +/- 0.03 for the switch sequence reported here. The change between the two states happens on a rapid kinetic time scale. A framework is introduced to aid in the design of AlloSwitches aimed at other targets. A high-affinity probe segment must be available to bind the target in the O-form, while a cover segment hides the probe in H. A key is adjusting the cover sequence to favor the H-form by a factor of 10-1000. This affords a robust response to small changes in target concentration, while saturation produces more than 90% of the maximal change in fluorescence. When a competitor displaces the switch from the NC-O complex, the released switch reverts to the H-form. This is the basis for a mix-and-read strategy for high-throughput screening of anti-nucleocapsid drug candidates that is much simpler to execute than traditional assays that require immobilization and washing steps.

Changes in drug 13C NMR chemical shifts as a tool for monitoring interactions with DNA.

The antibiotic drug, netropsin, was complexed with the DNA oligonucleotide duplex [d(GGTATACC)]2 to monitor drug 13C NMR chemical shifts changes. The binding mode of netropsin to the minor groove of DNA is well-known, and served as a good model for evaluating the relative sensitivity of 13C chemical shifts to hydrogen bonding. Large downfield shifts were observed for four resonances of carbons that neighbor sites which are known to form hydrogen bond interactions with the DNA minor groove. Many of the remaining resonances of netropsin exhibit shielding or relatively smaller deshielding changes. Based on the model system presented here, large deshielding NMR shift changes of a ligand upon macromolecule binding can likely be attributed to hydrogen bond formation at nearby sites.

Revised UV extinction coefficients for nucleoside-5'-monophosphates and unpaired DNA and RNA.

Ultraviolet absorption provides the nearly universal basis for determining concentrations of nucleic acids. Values for the UV extinction coefficients of DNA and RNA rely on the mononucleotide values determined 30-50 years ago. We show that nearly all of the previously published extinction coefficients for the nucleoside-5'-monophosphates are too large, and in error by as much as 7%. Concentrations based on complete hydrolysis and the older set of values are too low by approximately 4% for typical RNA and 2-3% for typical DNA samples. We also analyzed data in the literature for the extinction coefficients of unpaired DNA oligomers. Robust prediction of concentrations can be made using 38 microg/A260 unit for single-stranded DNA (ssDNA) having non-repetitive sequences and 40-80% GC. This is superior to currently used predictions that account for nearest-neighbor frequency or base composition. The latter result in concentrations that are 10-30% too low for typical ssDNA used as primers for PCR and other similar techniques. Methods are described here to accurately measure concentrations of nucleotides by nuclear magnetic resonance. NMR can be used to accurately determine concentrations (and extinction coefficients) of biomolecules within 1%.

Stem of SL1 RNA in HIV-1: structure and nucleocapsid protein binding for a 1 x 3 internal loop.

The 5'-leader of HIV-1 RNA controls many viral functions. Nucleocapsid (NC) domains of gag-precursor proteins select genomic RNA for packaging by binding several sites in the leader. One is likely to be a stem defect in SL1 that can adopt either a 1 x 3 internal loop, SL1i (including G247, A271, G272, G273) or a 1 x 1 internal loop (G247 x G273) near a two-base bulge (A269-G270). It is likely that these two conformations are both present and exchange readily. A 23mer RNA construct described here models SL1i and cannot slip into the alternate form. It forms a 1:1 complex with NCp7, which interacts most strongly at G247 and G272 (K(d) = 140 nM). This demonstrates that a linear G-X-G sequence is unnecessary for high-affinity binding. The NMR-based structure shows an easily broken G247:A271 base pair. G247 stacks on both of its immediate neighbors and A271 on its 5'-neighbor; G272 and G273 are partially ordered. A bend in the helix axis between the SL1 stems on either side of the internal loop is probable. An important step in maturation of the virus is the transition from an apical loop-loop interaction to a dimer involving intermolecular interactions along the full length of SL1. A bend in the stem may be important in relieving strain and ensuring that the strands do not become entangled during the transition. A stem defect with special affinity for NCp7 may accelerate the rate of the dimer transformation. This complex could become an important target for anti-HIV drug development, where a drug could exert its action near a high-energy intermediate on the pathway for maturation of the dimer.

Affinities of the nucleocapsid protein for variants of SL3 RNA in HIV-1.

Efficient packaging of genomic RNA into new HIV-1 virus particles requires that nucleocapsid domains of precursor proteins bind the SL3 tetraloop (G317-G-A-G320) from the 5'-untranslated region. This paper presents the affinities of 35 RNA variants of SL3 for the mature 55mer NC protein, as measured by fluorescence quenching of tryptophan-37 in the protein by nucleobases. The 1:1 complexes that form in 0.2 M NaCl have dissociation constants ranging from 8 nM (GGUG) to 20 microM (GAUA). The highly conserved (GGAG) sequence for the wild type is not the most stable (K(d) = 28 nM), suggesting that other selective pressures beyond the stability of the complex must be satisfied. The leading requirement for strong interaction is for G320, followed closely by G318. Replacing either with U, A, or C reduces affinity by a factor of 15-120. NC-domains from multiple proteins combine to recognize unpaired G(2)-loci, where two guanines are in close proximity. We have previously measured affinities of the NC protein for the important stem-loops of the major packaging domain [Shubsda, M. F., Paoletti, A. C., Hudson, B. S., and Borer, P. N. (2002) Biochemistry 41, 5276-82]. Comparison with the present work shows that the nature of the stem also modulates NC-RNA interactions. Placing the G(2)-loci from the apical SL2 or SL1 loops on the SL3 stem increases affinity by a factor of 2-3, while placing the SL4 loop on the SL3 stem reduces affinity 50-fold. These results are interesting in the context of RNA-protein interaction, as well as for the discovery of antiNC agents for AIDS therapy.

Affinities of packaging domain loops in HIV-1 RNA for the nucleocapsid protein.

To design anti-nucleocapsid drugs, it is useful to know the affinities the protein has for its natural substrates under physiological conditions. Dissociation equilibrium constants are reported for seven RNA stem-loops bound to the mature HIV-1 nucleocapsid protein, NCp7. The loops include SL1, SL2, SL3, and SL4 from the major packaging domain of genomic RNA. The binding assay is based on quenching the fluorescence of tryptophan-37 in the protein by G residues in the single-stranded loops. Tightly bound RNA molecules quench nearly all the fluorescence of freshly purified NCp7 in 0.2 M NaCl. In contrast, when the GGAG-tetraloop of tight-binding SL3 is replaced with UUCG or GAUA, quenching is almost nil, indicating very low affinity. Interpreting fluorescence titrations in terms of a rapidly equilibrating 1:1 complex explains nearly all of the experimental variance for the loops. Analyzed in this way, the highest affinities are for 20mer SL3 and 19mer SL2 hairpin constructs (K(d) = 28 +/- 3 and 23 +/- 2 nM, respectively). The 20mer stem-UUCG-loop and GAUA-loop constructs have <0.5% of the affinity for NCp7 relative to SL3. Affinities relative to SL3 for the other stem-loops are the following: 10% for a 16mer construct to model SL4, 30% for a 27mer model of the 9-residue apical loop of SL1, and 20% for a 23mer model of a 1 x 3 asymmetric internal loop in SL1. A 154mer construct that includes all four stem-loops binds tightly to NCp7, with the equivalent of three NCp7 molecules bound with high affinity per RNA; it is also possible that two strong sites and several weaker ones combine to give the appearance of three strong sites.