Thermodynamic criteria for high hit rate antisense oligonucleotide design.

Antisense oligonucleotides are used for therapeutic applications and in functional genomic studies. In practice, however, many of the oligonucleotides complementary to an mRNA have little or no antisense activity. Theoretical strategies to improve the 'hit rate' in antisense screens will reduce the cost of discovery and may lead to identification of antisense oligonucleotides with increased potency. Statistical analysis performed on data collected from more than 1000 experiments with phosphorothioate-modified oligonucleotides revealed that the oligo-probes, which form stable duplexes with RNA (DeltaG(o)37 < or = -30 kcal/mol) and have small self-interaction potential, are more frequently efficient than molecules that form less stable oligonucleotide-RNA hybrids or more stable self-structures. To achieve optimal statistical preference, the values for self-interaction should be (DeltaG(o)37) > or = -8 kcal/mol for inter-oligonucleotide pairing and (DeltaG(o)37) > or = -1.1 kcal/mol for intra-molecular pairing. Selection of oligonucleotides with these thermodynamic values in the analyzed experiments would have increased the 'hit rate' by as much as 6-fold.

Fully modified 2' MOE oligonucleotides redirect polyadenylation.

Many genes have been described and characterized that have alternative polyadenylation signals at the 3'-end of their pre-mRNAs. Many of these same messages also contain destabilization motifs responsible for rapid degradation of the mRNA. Polyadenylation site selection can thus determine the stability of an mRNA. Fully modified 2'-O:-methoxy ethyl/phosphorothioate oligonucleotides that hybridize to the 3'-most polyadenylation site or signal of E-selectin were able to inhibit polyadenylation at this site and redirect it to one of two upstream cryptic sites. The shorter transcripts produced after antisense treatment have fewer destabilization sequences, increased mRNA stability and altered protein expression. This study demonstrates that antisense oligonucleotides can be successfully employed to redirect polyadenylation. This is the first demonstration of the use of oligonucleotides to increase, rather than decrease, abundance of a message.

Pharmacokinetic properties of 2'-O-(2-methoxyethyl)-modified oligonucleotide analogs in rats.

Plasma pharmacokinetics, biodistribution, excretion, and metabolism of four modified 20-mer antisense oligonucleotides targeted to human intercellular adhesion molecule-1 mRNA have been characterized in rats and compared with a first-generation phosphorothioate oligodeoxynucleotide (PS ODN), ISIS 2302. The modified oligonucleotides contained 2'-O-(2-methoxyethyl) (2'-O-MOE) ribose sugar modifications on all or a portion of the nucleotides in the antisense sequence. The 2'-O-MOE-modified oligonucleotides were resistant to nuclease metabolism in both plasma and tissue. In general, plasma pharmacokinetics was not substantially altered by addition of the 2'-O-MOE modification to PS ODN. Thus, plasma clearance was dominated by distribution to tissues, broadly, with less than 10% of the administered dose excreted in urine or feces over 24 h. However, the 2'-O-MOE modification combined with the phosphodiester (PO) backbone exhibited 10-fold more rapid plasma clearance, with approximately 50% of the dose excreted in urine as intact oligonucleotide. Consistent with its rapid and extensive excretion, the PO 2'-O-MOE modification distributed to very few organs in any substantial amount with the exception of the kidney. Oligonucleotides that contained phosphorothioate backbones were highly bound to plasma proteins. Indeed, the primary characteristic that resulted in the most marked alterations in pharmacokinetics appeared to be the affinity and capacity of these compounds to bind plasma proteins. A balance of greater stability supplied by the 2'-O-MOE modification together with maintenance of plasma protein binding appears to be necessary to ensure favorable pharmacokinetics of this new generation of antisense oligonucleotides.

Identification of sequence motifs in oligonucleotides whose presence is correlated with antisense activity.

Design of antisense oligonucleotides targeting any mRNA can be much more efficient when several activity-enhancing motifs are included and activity-decreasing motifs are avoided. This conclusion was made after statistical analysis of data collected from >1000 experiments with phosphorothioate-modified oligonucleotides. Highly significant positive correlation between the presence of motifs CCAC, TCCC, ACTC, GCCA and CTCT in the oligonucleotide and its antisense efficiency was demonstrated. In addition, negative correlation was revealed for the motifs GGGG, ACTG, AAA and TAA. It was found that the likelihood of activity of an oligonucleotide against a desired mRNA target is sequence motif content dependent.

Patterns of variant polyadenylation signal usage in human genes.

The formation of mature mRNAs in vertebrates involves the cleavage and polyadenylation of the pre-mRNA, 10-30 nt downstream of an AAUAAA or AUUAAA signal sequence. The extensive cDNA data now available shows that these hexamers are not strictly conserved. In order to identify variant polyadenylation signals on a large scale, we compared over 8700 human 3' untranslated sequences to 157,775 polyadenylated expressed sequence tags (ESTs), used as markers of actual mRNA 3' ends. About 5600 EST-supported putative mRNA 3' ends were collected and analyzed for significant hexameric sequences. Known polyadenylation signals were found in only 73% of the 3' fragments. Ten single-base variants of the AAUAAA sequence were identified with a highly significant occurrence rate, potentially representing 14.9% of the actual polyadenylation signals. Of the mRNAs, 28.6% displayed two or more polyadenylation sites. In these mRNAs, the poly(A) sites proximal to the coding sequence tend to use variant signals more often, while the 3'-most site tends to use a canonical signal. The average number of ESTs associated with each signal type suggests that variant signals (including the common AUUAAA) are processed less efficiently than the canonical signal and could therefore be selected for regulatory purposes. However, the position of the site in the untranslated region may also play a role in polyadenylation rate.

Effects of RNA secondary structure on cellular antisense activity.

The secondary and tertiary structures of a mRNA are known to effect hybridization efficiency and potency of antisense oligonucleotides in vitro. Additional factors including oligonucleotide stability and cellular uptake are also thought to contribute to antisense potency in vivo. Each of these factors can be affected by the sequence of the oligonucleotide. Although mRNA structure is presumed to be a critical determinant of antisense activity in cells, to date little direct experimental evidence has addressed the significance of structure. In order to determine the importance of mRNA structure on antisense activity, oligonucleotide target sites were cloned into a luciferase reporter gene along with adjoining sequence to form known structures. This allowed us to study the effect of target secondary structure on oligonucleotide binding in the cellular environment without changing the sequence of the oligonucleotide. Our results show that structure does play a significant role in determining oligonucleotide efficacy in vivo. We also show that potency of oligonucleotides can be improved by altering chemistry to increase affinity for the mRNA target even in a region that is highly structured.

Predicting oligonucleotide affinity to nucleic acid targets.

A computer program, OligoWalk, is reported that predicts the equilibrium affinity of complementary DNA or RNA oligonucleotides to an RNA target. This program considers the predicted stability of the oligonucleotide-target helix and the competition with predicted secondary structure of both the target and the oligonucleotide. Both unimolecular and bimolecular oligonucleotide self structure are considered with a user-defined concentration. The application of OligoWalk is illustrated with three comparisons to experimental results drawn from the literature.

Expression of the insulin-like growth factors and their receptors in adenocarcinoma of the colon.

AIMS: To study changes in the expression of insulin-like growth factors (IGFs) and their receptors, as well as production of the IGF-I and IGF-II polypeptides, in adenocarcinoma of the colon. METHODS: Malignant tissue obtained at operation was used. Total RNA was extracted and specific IGF-I and IGF-II and their receptor mRNAs were measured by a solution hybridisation RNase protection assay. IGF-I and IGF-II polypeptides were measured by specific immunoassays. RESULTS: All normal tissues expressed IGF-II, IGF-I receptor, and IGF-II/mannose-6-phosphate (Man-6-P) receptor. IGF-I mRNA could not be detected but the polypeptide was present in small but equal amounts in normal and malignant tissue. IGF-II was expressed 40 times more abundantly in colonic tumours than in adjacent normal tissue and the concentration of the corresponding polypeptide was twice as high in the malignant tissue. IGF-I receptor expression was increased by a factor of 2.5 and IGF-II/Man-6-P receptor by a factor of 4. CONCLUSIONS: This study confirms that in adenocarcinoma of the human colon there is increased expression of IGF-I receptor and IGF-II. Furthermore, IGF-II/Man-6-P receptor message is increased and the increase in IGF-II message is accompanied by a doubling of the IGF-II protein in the tumour tissue compared with the adjacent normal tissue. These findings suggest that the IGF-II/Man-6-P receptor may also be involved in development of adenocarcinoma of the colon. There is rapidly accumulating evidence implicating the IGF system in the development of malignancy of the large bowel.

Are intraepithelial lymphocytes in celiac mucosa responsible for inducing programmed cell death (apoptosis) in enterocytes? Histochemical demonstration of perforins in cytoplasmic granules of intraepithelial lymphocytes.

BACKGROUND: Programmed cell death refers to the genetically determined processes by which cells die in response to physiologic extracellular and intracellular signals, morphologically described as apoptosis. In physiologic and pathologic circumstances this process may involve effector and target cells. METHODS: To identify serine esterase granules in intraepithelial lymphocytes, fresh-frozen human small intestine mucosal sections from normal and celiac-affected mucosa were incubated with substrate-specific N-alpha-benzyloxy-carbonyl-L-lysine thiobenzyl (BLT) and a chromogen (4 Benzoylamino-2,5-diethoxybenzene-dazonium chloride hemi [zinc chloride] salt as capture agent and were examined by light microscopy. RESULTS: Normal mucosa showed an occasional intraepithelial lymphocyte with BLT-positive intracytoplasmic granules. Some large mononuclear cells of the lamina propria were similarly stained. Many more intraepithelial lymphocytes were BLT-positive among the surface enterocytes of untreated celiac mucosa. Lamina propria mononuclear cells close to the basal layer of crypt cells also appeared to be increased. CONCLUSIONS: The histochemical identification of BLT-positive esters within intraepithelial lymphocytes suggests their involvement in enterocyte death under physiologic conditions. The increased BLT-positive intraepithelial lymphocytes found in the celiac mucosa may be related to the known increase in cytotoxic intraepithelial lymphocytes in untreated celiac disease.

Histologic evaluation of endoscopic versus suction biopsies of small intestinal mucosae in children with and without celiac disease.

BACKGROUND: Concern over the adequacy of histologic diagnosis of endoscopic duodenal biopsies in children prompted this comparative study on the histologic quality of endoscopic versus capsule biopsies. We found this problem addressed in only six previous reports. METHODS: Blind examinations of the histologic sections of 48 duodenal biopsies obtained by gastrointestinal endoscopy in children aged 2-18 years were compared to 52 biopsies obtained by the small bowel suction method (from children aged 1-16 years). RESULTS: Although 87.5% of endoscopic biopsies and 94.2% of capsule biopsies were adequate for histologic diagnosis, fragmentation or squashing was seen in 83.3% of endoscopic biopsies and only in 25% of capsule biopsies. CONCLUSIONS: Biopsies obtained by suction are of better quality than those obtained by endoscopy. If endoscopy is preferred for technical reasons, the following conditions should be observed: the patients should be aged over 2 years, and a minimum of four biopsies should be obtained with forceps of a diameter greater than 2 mm. Adequate histologic criteria for diagnosis should include at least one full-thickness mucosal specimen more than 3 mm in length, vertically oriented, and not fragmented. In children under age 2, duodenal or jejunal capsule biopsies are preferred, since the specimens are usually larger and less fragmented. Endoscopy is technically more difficult in the very young patient.

Hereditary CD4+ T lymphocytopenia.

Two siblings suffering from mental retardation, progressive bronchiectasis, extensive warts, and persistent hepatitis B are described. The propositus also had an unusual physiognomy and non-specific colitis. Both patients had a marked decrease in the population of CD4+ helper T cells.

What affects the effect of 2'-alkoxy modifications? 1. Stabilization effect of 2'-methoxy substitutions in uniformly modified DNA oligonucleotides.

The thermostability of hybrid duplexes with uniformly 2'-methoxy modified DNA strands (D'R and RD'), their unmodified DNA:RNA counterparts (DR and RD), and corresponded RNA:RNA (RR) duplexes for six sequences with different GC and deoxypyrimidine (dPy) content was measured. The linear correlation between the total stabilization effect of 2'-methoxy modifications (Delta DeltaG(o)37(D'R-DR)) and the relative stability of corresponding unmodified hybrids compared to the RR counterparts (Delta DeltaG(o)37(RR-DR)) suggests that the initial conformational and the thermodynamic state of the "parent" unmodified hybrid governs the effect of 2'-methoxy (and may be other 2'-alkoxy) modifications whose mechanism of action includes an S --> N conformational shift resulting in an RNA-like A-form duplex. We also found a correlation between the "hydrophobic" part of the total effect (Delta DeltaG(o)37(D'R-RR)) and the dA fraction in the modified DNA strand, suggesting that the "hydrophobic" effect of the 2'-methoxy groups results mainly from intraresidue steric effects increasing rigidity of the modified sugar rings. The correlations observed enabled us to predict the stability of hybrids with 2'-methoxy modified DNA strands for any sequence except for sequences with (dU)10 and (dA)10 strings.

Evaluation of pyrimidine PNA binding to ssDNA targets from nonequilibrium melting experiments.

Slow kinetics of homopyrimidine PNA binding to single stranded DNA and RNA targets is manifested in significant hysteresis in thermal UV absorption experiments. We have compared temperatures of dissociation (Tdis) and reassociation (Tass) for triplexes formed by DNA and single or bis PNAs with K50 derived from gel mobility experiments. Results indicated there was no correlation between Tdis and K50 while reasonable correlation between Tass and K50 was found. This correlation enabled use of easy thermal UV absorption experiments for evaluation of PNA binding to DNA/RNA targets.

Complex FISH probes for the subtelomeric regions of all human chromosomes: comparative hybridization of CEPH YACs to chromosomes of the Old World monkey Presbytis cristata and great apes.

We have generated a human subtelomere probe panel, utilizing well characterized CEPH YACs, for the investigation of human chromosome pathology and evolution through fluorescent in situ hybridization (FISH). Region-specific FISH probes will be extremely valuable for detecting cytogenetically cryptic telomere abnormalities. Here, we present the first comparative mapping study (with 29 subtelomere probes and 6 chromosome paints) to the Old World monkey Presbytis cristata, followed by hybridizations to the great apes, gorilla and orangutan, when rearrangements were detected. We observed that the position of telomere-associated genomic sequences has been only moderately conserved during primate evolution. YAC 364f9, specific for the subtelomeric long arm of human chromosome 3, contains an evolutionary inversion breakpoint that was involved in independent chromosome rearrangements in P. cristata and gorilla.

The ups and downs of nucleic acid duplex stability: structure-stability studies on chemically-modified DNA:RNA duplexes.

In an effort to discover novel oligonucleotide modifications for antisense therapeutics, we have prepared oligodeoxyribonucleotides containing more than 200 different modifications and measured their affinities for complementary RNA. These include modifications to the heterocyclic bases, the deoxy-ribose sugar and the phosphodiester linkage. From these results, we have been able to determine structure-activity relationships that correlate hybridization affinity with changes in oligonucleotide structure. Data for oligonucleotides containing modified pyrimidine nucleotides are presented. In general, modifications that resulted in the most stable duplexes contained a heteroatom at the 2'-position of the sugar. Other sugar modifications usually led to diminished hybrid stability. Most backbone modifications that led to improved hybridization restricted backbone mobility and resulted in an A-type sugar pucker for the residue 5'to the modified internucleotide linkage. Among the heterocycles, C-5-substituted pyrimidines stood out as substantially increasing duplex stability.

Strategies for rapid deconvolution of combinational libraries: comparative evaluation using a model system.

Synthesis and testing of complex mixtures maximize the number of compounds that can be prepared and tested in a combinatorial library. When mixtures of compounds are screened, however, the identity of the compound(s) selected may depend on the deconvolution procedure employed. Previously, we developed a model system for evaluation of deconvolution procedures and used it to compare pooling strategies for iterative and noniterative deconvolution [Freier et al. J. Med. Chem. 1995, 38, 344-352]. We have now extended the model studies to include simulations of procedures with overlapping subsets such as subtractive pooling [Carell et al. Angew, Chem., Int. Ed. Engl. 1994, 33, 2061-2064], bogus coin pooling [Blake and Litzi-Davis. Bioconjugate Chem. 1992, 3, 510-513], and orthogonal pooling [D'Prez et al. J. Am. Chem. Soc. 1995, 117, 5405-5406]. These strategies required synthesis and testing of fewer subsets than did the more traditional nonoverlapping iterative strategies. The compounds identified using simulations of these strategies, however, were not the most active compounds in the library and were substantially less active than those identified by simulations of more traditional strategies.