OSIRIS-REx Contamination Control Strategy and Implementation.

OSIRIS-REx will return pristine samples of carbonaceous asteroid Bennu. This article describes how pristine was defined based on expectations of Bennu and on a realistic understanding of what is achievable with a constrained schedule and budget, and how that definition flowed to requirements and implementation. To return a pristine sample, the OSIRIS-REx spacecraft sampling hardware was maintained at level 100 A/2 and <180 ng/cm2 of amino acids and hydrazine on the sampler head through precision cleaning, control of materials, and vigilance. Contamination is further characterized via witness material exposed to the spacecraft assembly and testing environment as well as in space. This characterization provided knowledge of the expected background and will be used in conjunction with archived spacecraft components for comparison with the samples when they are delivered to Earth for analysis. Most of all, the cleanliness of the OSIRIS-REx spacecraft was achieved through communication among scientists, engineers, managers, and technicians.

Blocking transcription of the human rhodopsin gene by triplex-mediated DNA photocrosslinking.

To explore the ability of triplex-forming oligodeoxyribonucleotides (TFOs) to inhibit genes responsible for dominant genetic disorders, we used two TFOs to block expression of the human rhodopsin gene, which encodes a G protein-coupled receptor involved in the blinding disorder autosomal dominant retinitis pigmentosa. Psoralen-modified TFOs and UVA irradiation were used to form photoadducts at two target sites in a plasmid expressing a rhodopsin-EGFP fusion, which was then transfected into HT1080 cells. Each TFO reduced rhodopsin-GFP expression by 70-80%, whereas treatment with both reduced expression by 90%. Expression levels of control genes on either the same plasmid or one co-transfected were not affected by the treatment. Mutations at one TFO target eliminated its effect on transcription, without diminishing inhibition by the other TFO. Northern blots indicated that TFO-directed psoralen photoadducts blocked progression of RNA polymerase, resulting in truncated transcripts. Inhibition of gene expression was not relieved over a 72 h period, suggesting that TFO-induced psoralen lesions are not repaired on this time scale. Irradiation of cells after transfection with plasmid and psoralen-TFOs produced photoadducts inside the cells and also inhibited expression of rhodopsin-EGFP. We conclude that directing DNA damage with psoralen-TFOs is an efficient and specific means for blocking transcription from the human rhodopsin gene.

Role of the nucleotide excision repair gene ERCC1 in formation of recombination-dependent rearrangements in mammalian cells.

Spontaneous recombination between direct repeats at the adenine phosphoribosyltransferase (APRT) locus in ERCC1-deficient cells generates a high frequency of rearrangements that are dependent on the process of homologous recombination, suggesting that rearrangements are formed by misprocessing of recombination intermediates. Given the specificity of the structure-specific Ercc1/Xpf endonuclease, two potential recombination intermediates are substrates for misprocessing in ERCC1(-) cells: heteroduplex loops and heteroduplex intermediates with non-homologous 3' tails. To investigate the roles of each, we constructed repeats that would yield no heteroduplex loops during spontaneous recombination or that would yield two non-homologous 3' tails after treatment with the rare-cutting endonuclease I-SCE:I. Our results indicate that misprocessing of heteroduplex loops is not the major source of recombination-dependent rearrangements in ERCC1-deficient cells. Our results also suggest that the Ercc1/Xpf endonuclease is required for efficient removal of non-homologous 3' tails, like its Rad1/Rad10 counterpart in yeast. Thus, it is likely that misprocessing of non-homologous 3' tails is the primary source of recombination-dependent rearrangements in mammalian cells. We also find an unexpected effect of ERCC1 deficiency on I-SCE:I-stimulated rearrangements, which are not dependent on homologous recombination, suggesting that the ERCC1 gene product may play a role in generating the rearrangements that arise after I-SCE:I-induced double-strand breaks.

Psoralen photo-cross-linking by triplex-forming oligonucleotides at multiple sites in the human rhodopsin gene.

Targeting DNA damage by triplex-forming oligonucleotides (TFOs) represents a way of modifying gene expression and structure and a possible approach to gene therapy. We have determined that this approach can deliver damage with great specificity to sites in the human gene for the G-protein-linked receptor rhodopsin, mutations of which can lead to the genetic disorder autosomal dominant retinitis pigmentosa. We have introduced DNA monoadducts and interstrand cross-links at multiple target sites within the gene using TFOs with a photoactivatable psoralen group at the 5'-end. The extent of formation of photoadducts (i.e., monoadducts and cross-links) was measured at target sites with a 5'-ApT sequence at the triplex-duplex junction and at a target site with 5'-ApT and 5'-TpA sequences located four and seven nucleotides away, respectively. To improve psoralen reactivity at more distant sites, psoralen moieties were attached to TFOs with nucleotide "linkers" from two to nine nucleotides in length. High-affinity binding was maintained with linkers of up to 10 nucleotides, but affinities tended to decrease somewhat with increasing linker length due to faster dissociation kinetics. DNase I footprinting indicated little, if any, interaction between linkers and the duplex. Psoralen-TFO conjugates formed DNA cross-links with high efficiency (56-65%) at 5'-ApT sequences located at triplex junctions. At a 5'-ApT site four nucleotides away, the efficiency varied with linker length; a four-nucleotide linker gave the highest efficiency. Duplexes with 5'-TpA and 5'-ApT sites two nucleotides away, in otherwise identical sequences, were cross-linked with efficiencies of 56 and 38%, respectively. These results indicate that TFO-linker-psoralen conjugates allow simultaneous, efficient targeting of multiple sites in the human rhodopsin gene.

Triplex targets in the human rhodopsin gene.

We have explored the application of triplex technology to the human rhodopsin gene, which encodes a G-protein-linked receptor involved in the genetic disorder autosomal dominant retinitis pigmentosa (ADRP). Our results support the hypothesis that most human genes contain high-affinity triplex sites and further refine the rules governing identification and successful targeting of triplex-forming oligonucleotides (TFOs) to these sites. Using a computer search for sites 15 nucleotides in length and greater than 80% purine, we found 143 distinct sites in the rhodopsin gene and comparable numbers of sites in several other human genes. By applying more stringent criteria, we selected 17 potential target sites in the rhodopsin gene, screened them with a plasmid binding assay, and found 8 that bound TFOs with submicromolar affinity (Kd = 10(-)9-10(-)7 M). We compared purine (GA) and mixed (GT) TFOs at each site, and found that GA-TFOs consistently bound with higher affinity, and were less sensitive to pyrimidine interruptions in the target strand. High G-content favored high-affinity binding; only sites with >54% G-content bound TFOs with Kd