Hybridization Affinity Research Articles

DNA surface hybridization regimes

Surface hybridization reactions, in which sequence-specific recognition occurs between immobilized and solution nucleic acids, are routinely carried out to quantify and interpret genomic information. Although hybridization is fairly well understood in bulk solution, the greater complexity of an interfacial environment presents new challenges to a fundamental understanding, and hence application, of these assays. At a surface, molecular interactions are amplified by the two-dimensional nature of the immobilized layer, which focuses the nucleic acid charge and concentration to levels not encountered in solution, and which impacts the hybridization behavior in unique ways. This study finds that, at low ionic strengths, an electrostatic balance between the concentration of immobilized oligonucleotide charge and solution ionic strength governs the onset of hybridization. As ionic strength increases, the importance of electrostatics diminishes and the hybridization behavior becomes more complex. Suppression of hybridization affinity constants relative to solution values, and their weakened dependence on the concentration of DNA counterions, indicate that the immobilized strands form complexes that compete with hybridization to analyte strands. Moreover, an unusual regime is observed in which the surface coverage of immobilized oligonucleotides does not significantly influence the hybridization behavior, despite physical closeness and hence compulsory interactions between sites. These results are interpreted and summarized in a diagram of hybridization regimes that maps specific behaviors to experimental ranges of ionic strength and probe coverage.

Synthesis and hybridization properties of 2′- O-(tetrazol-5-yl)ethyl-modified oligonucleotides

2′- O-(1 H-Tetrazol-5-yl)ethyladenosine was synthesized using 2′- O-cyanoethyladenosine derivative as a key intermediate. The 2′- O-(1 H-tetrazol-5-yl)ethyl modifications exhibited intriguing properties such as the change in the structure of the tetrazole residue between a protonated and a deprotonated form. The T m experiments of various oligodeoxynucleotides having a 2′- O-(1 H-tetrazol-5-yl)ethyl-modified adenosine showed reduced hybridization affinity in comparison to the unmodified oligonucleotides toward their complementary oligodeoxynucleotides. The mechanism of the reduced hybridization affinity was discussed on the basis of the structure and the physicochemical properties of the tetrazole moiety.

‘Protected DNA Probes’ capable of strong hybridization without removal of base protecting groups

We propose a new strategy called the ‘Protected DNA Probes (PDP) method’ in which appropriately protected bases selectively bind to the complementary bases without the removal of their base protecting groups. Previously, we reported that 4-N-acetylcytosine oligonucleotides (ac4C) exhibited a higher hybridization affinity for ssDNA than the unmodified oligonucleotides. For the PDP strategy, we created a modified adenine base and synthesized an N-acylated deoxyadenosine mimic having 6-N-acetyl-8-aza-7-deazaadenine (ac6az8c7A). It was found that PDP containing ac4C and ac6az8c7A exhibited higher affinity for the complementary ssDNA than the corresponding unmodified DNA probes and showed similar base recognition ability. Moreover, it should be noted that this PDP strategy could guarantee highly efficient synthesis of DNA probes on controlled pore glass (CPG) with high purity and thereby could eliminate the time-consuming procedures for isolating DNA probes. This strategy could also avoid undesired base-mediated elimination of DNA probes from CPG under basic conditions such as concentrated ammonia solution prescribed for removal of base protecting groups in the previous standard approach. Here, several successful applications of this strategy to single nucleotide polymorphism detection are also described in detail using PDPs immobilized on glass plates and those prepared on CPG plates, suggesting its potential usefulness.

Impact of point-mutations on the hybridization affinity of surface-bound DNA/DNA and RNA/DNA oligonucleotide-duplexes: Comparison of single base mismatches and base bulges

BackgroundThe high binding specificity of short 10 to 30 mer oligonucleotide probes enables single base mismatch (MM) discrimination and thus provides the basis for genotyping and resequencing microarray applications. Recent experiments indicate that the underlying principles governing DNA microarray hybridization – and in particular MM discrimination – are not completely understood. Microarrays usually address complex mixtures of DNA targets. In order to reduce the level of complexity and to study the problem of surface-based hybridization with point defects in more detail, we performed array based hybridization experiments in well controlled and simple situations.ResultsWe performed microarray hybridization experiments with short 16 to 40 mer target and probe lengths (in situations without competitive hybridization) in order to systematically investigate the impact of point-mutations – varying defect type and position – on the oligonucleotide duplex binding affinity. The influence of single base bulges and single base MMs depends predominantly on position – it is largest in the middle of the strand. The position-dependent influence of base bulges is very similar to that of single base MMs, however certain bulges give rise to an unexpectedly high binding affinity. Besides the defect (MM or bulge) type, which is the second contribution in importance to hybridization affinity, there is also a sequence dependence, which extends beyond the defect next-neighbor and which is difficult to quantify. Direct comparison between binding affinities of DNA/DNA and RNA/DNA duplexes shows, that RNA/DNA purine-purine MMs are more discriminating than corresponding DNA/DNA MMs. In DNA/DNA MM discrimination the affected base pair (C·G vs. A·T) is the pertinent parameter. We attribute these differences to the different structures of the duplexes (A vs. B form).ConclusionWe have shown that DNA microarrays can resolve even subtle changes in hybridization affinity for simple target mixtures. We have further shown that the impact of point defects on oligonucleotide stability can be broken down to a hierarchy of effects. In order to explain our observations we propose DNA molecular dynamics – in form of zipping of the oligonucleotide duplex – to play an important role.

Efficient siRNA selection using hybridization thermodynamics

Small interfering RNA (siRNA) are widely used to infer gene function. Here, insights in the equilibrium of siRNA-target hybridization are used for selection of efficient siRNA. The accessibilities of siRNA and target mRNA for hybridization, as measured by folding free energy change, are shown to be significantly correlated with efficacy. For this study, a partition function calculation that considers all possible secondary structures is used to predict target site accessibility; a significant improvement over calculations that consider only the predicted lowest free energy structure or a set of low free energy structures. The predicted thermodynamic features, in addition to siRNA sequence features, are used as input for a support vector machine that selects functional siRNA. The method works well for predicting efficient siRNA (efficacy >70%) in a large siRNA data set from Novartis. The positive predictive value (percentage of sites predicted to be efficient for silencing that are) is as high as 87.6%. The sensitivity and specificity are 22.7 and 96.5%, respectively. When tested on data from different sources, the positive predictive value increased 8.1% by adding equilibrium terms to 25 local sequence features. Prediction of hybridization affinity using partition functions is now available in the RNAstructure software package.

Splicing Segregation: The Minor Spliceosome Acts outside the Nucleus and Controls Cell Proliferation

The functional relevance and the evolution of two parallel mRNA splicing systems in eukaryotes--a major and minor spliceosome that differ in abundance and splicing rate--are poorly understood. We report here that partially spliced pre-mRNAs containing minor-class introns undergo nuclear export and that minor-class snRNAs are predominantly cytoplasmic in vertebrates. Cytoplasmic interference with the minor spliceosome further indicated its functional segregation from the nucleus. In keeping with this, minor splicing was only weakly affected during mitosis. By selectively interfering with snRNA function in zebrafish development and in mammalian cells, we revealed a conserved role for minor splicing in cell-cycle progression. We argue that the segregation of the splicing systems allows for processing of partially unspliced cytoplasmic transcripts, emerging as a result of different splicing rates. The segregation offers a mechanism accounting for spliceosome evolution in a single lineage and provides a means for nucleus-independent control of gene expression.

Prion protein aggresomes are poly(A) + ribonucleoprotein complexes that induce a PKR-mediated deficient cell stress response

In mammalian cells, cytoplasmic protein aggregates generally coalesce to form aggresomal particles. Recent studies indicate that prion-infected cells produce prion protein (PrP) aggresomes, and that such aggregates may be present in the brain of infected mice. The molecular activity of PrP aggresomes has not been fully investigated. We report that PrP aggresomes initiate a cell stress response by activating the RNA-dependent protein kinase (PKR). Activated PKR phosphorylates the translation initiation factor eIF2α, resulting in protein synthesis shut-off. However, other components of the stress response, including the assembly of poly(A) + RNA-containing stress granules and the synthesis of heat shock protein 70, are repressed. In situ hybridization experiments and affinity chromatography on oligo(dT)-cellulose showed that PrP aggresomes bind poly(A) + RNA, and are therefore poly(A) + ribonucleoprotein complexes. These findings support a model in which PrP aggresomes send neuronal cells into untimely demise by modifying the cell stress response, and by inducing the aggregation of poly(A) + RNA.

Phosphorothioate oligodeoxynucleotides: large-scale synthesis and analysis, impurity characterization, and the effects of phosphorus stereochemistry.

Large-scale synthesis of phosphorothioate oligodeoxynucleotides on Tentagel using a 'batch mode' synthesizer and beta-cyanoethyl phosphoramidite coupling followed by sulfurization with bis(O,O-diisopropoxy phosphinothioyl) disulfide (S-tetra) provides stepwise yields of 98-99% and results in phosphorothioate oligodeoxynucleotides that are 93-97% pure, as determined by PAGE, after reverse-phase high performance liquid chromatography (RP-HPLC) and 'downstream' processing. The purity of phosphorothioate oligodeoxynucleotides synthesized on Tentagel is significantly higher than those synthesized on controlled pore glass. Electrospray ionization mass spectrometry of the n-1 impurity isolated by preparative PAGE was used to establish that the n-1 impurity is a heterogeneous mixture of all possible single-deletion sequences, relative to the parent phosphorothioate oligodeoxynucleotide, and results from minor, though repetitive, imperfections in the synthesis cycle. Acid-catalysed depurination was found to occur both during the synthesis and during the post-synthesis detritylation, following RP-HPLC. Studies of hybridization affinity and biological mechanism of action using independently synthesized n-1 phosphorothioate oligodeoxynucleotides relative to the 15 mer LR-3280 showed that, in this case, the majority of the n-1 sequences had more than a 10 degrees C decrease in melting temperature with sense RNA compared to the n-mer, and they did not cause detectable cleavage of RNA by RNase H in HL-60 human promyelocytic leukaemia cells. P stereoregular phosphorothioate oligodeoxynucleotides are not significantly more active than their stereorandom counterparts and thus their use in clinical studies seems unwarranted.

Characterization of insulin/IGF hybrid receptors: contributions of the insulin receptor L2 and Fn1 domains and the alternatively spliced exon 11 sequence to ligand binding and receptor activation

The IR (insulin receptor) and IGFR (type I insulin-like growth factor receptor) are found as homodimers, but the respective pro-receptors can also heterodimerize to form insulin-IGF hybrid receptors. There are conflicting data on the ligand affinity of hybrids, and especially on the influence of different IR isoforms. To investigate further the contribution of individual ligand binding epitopes to affinity and specificity in the IR/IGFR family, we generated hybrids incorporating both IR isoforms (A and B) and IR/IGFR domain-swap chimaeras, by ectopic co-expression of receptor constructs in Chinese hamster ovary cells, and studied ligand binding using both radioligand competition and bioluminescence resonance energy transfer assays. We found that IR-A-IGFR and IR-B-IGFR hybrids bound insulin with similar relatively low affinity, which was intermediate between that of homodimeric IR and homodimeric IGFR. However, both IR-A-IGFR and IR-B-IGFR hybrids bound IGF-I and IGF-II with high affinity, at a level comparable with homodimeric IGFR. Incorporation of a significant fraction of either IR-A or IR-B into hybrids resulted in abrogation of insulin- but not IGF-I-stimulated autophosphorylation. We conclude that the sequence of 12 amino acids encoded by exon 11 of the IR gene has little or no effect on ligand binding and activation of IR-IGFR hybrids, and that hybrid receptors bind IGFs but not insulin at physiological concentrations regardless of the IR isoform they contained. To reconstitute high affinity insulin binding within a hybrid receptor, chimaeras in which the IGFR L1 or L2 domains had been replaced by equivalent IR domains were co-expressed with full-length IR-A or IR-B. In the context of an IR-A-IGFR hybrid, replacement of IR residues 325-524 (containing the L2 domain and part of the first fibronectin domain) with the corresponding IGFR sequence increased the affinity for insulin by 20-fold. We conclude that the L2 and/or first fibronectin domains of IR contribute in trans with the L1 domain to create a high affinity insulin-binding site within a dimeric receptor.

The Chemical and Biochemical Properties of Methylphosphotriester DNA and RNA in Comparison with Their Corresponding Methylphosphonates. A Dynamic Model Description

Methylphosphotriester DNA and RNA are of great interest to investigate their hybridization affinity with natural DNA and RNA with respect to their physical and biological properties. The results are compared with related modified oligonucleotides. Specific attention will be given to the development of recent antiretroviral nucleosides focused on their molecular conformation and the mechanistic aspects based on the physical properties of phosphorus in a trigonal bipyramidal configuration corresponding with in vitro and in vivo kinetics.

Hybrid homology modeling and mutational analysis of cytochrome P450C24A1 (CYP24A1) of the Vitamin D pathway: Insights into substrate specificity and membrane bound structure–function

Cytochrome P450C24A1 (CYP24A1), a peripheral inner mitochondrial membrane hemoprotein and candidate oncogene, regulates the side-chain metabolism and biological function of vitamin D and many of its related analog drugs. Rational mutational analysis of rat CYP24A1 based on hybrid (2C5/BM-3) homology modeling and affinity labeling studies clarified the role of key domains (N-terminus, A′, A, and F-helices, β3a strand, and β5 hairpin) in substrate binding and catalysis. The scope of our study was limited by an inability to purify stable mutant enzyme targeting soluble domains (B′, G, and I-helices) and suggested greater conformational flexibility among CYP24A1’s membrane-associated domains. The most notable mutants developed by modeling were V391T and I500A, which displayed defective-binding function and profound metabolic defects for 25-hydroxylated vitamin D3 substrates similar to a non-functional F-helix mutant (F249T) that we previously reported. Val-391 (β3a strand) and Ile-500 (β5 hairpin) are modeled to interact with Phe-249 (F-helix) in a hydrophobic cluster that directs substrate-binding events through interactions with the vitamin D cis-triene moiety. Prior affinity labeling studies identified an amino-terminal residue (Ser-57) as a putative active-site residue that interacts with the 3β-OH group of the vitamin D A-ring. Studies with 3-epi and 3-deoxy-1,25(OH)2D3 analogs confirmed interactions between the 3β-OH group and Ser-57 effect substrate recognition and trafficking while establishing that the trans conformation of A-ring hydroxyl groups (1α and 3β) is obligate for high-affinity binding to rat CYP24A1. Our work suggests that CYP24A1’s amphipathic nature allows for monotopic membrane insertion, whereby a pw2d-like substrate access channel is formed to shuttle secosteroid substrate from the membrane to the active-site. We hypothesize that CYP24A1 has evolved a unique amino-terminal membrane-binding motif that contributes to substrate specificity and docking through coordinated interactions with the vitamin D A-ring.

EphB3 receptor and ligand expression in the adult rat brain

Eph receptors and ligands are two families of proteins that control axonal guidance during development. Their expression was originally thought to be developmentally regulated but recent work has shown that several EphA receptors are expressed postnatally. The EphB3 receptors are expressed during embryonic development in multiple regions of the central nervous system but their potential expression and functional role in the adult brain is unknown. We used in situ hybridization, immunohistochemistry, and receptor affinity probe in situ staining to investigate EphB3 receptors mRNA, protein, and ligand (ephrin-B) expression, respectively, in the adult rat brain. Our results indicate that EphB3 receptor mRNA and protein are constitutively expressed in discrete regions of the adult rat brain including the cerebellum, raphe pallidus, hippocampus, entorhinal cortex, and both motor and sensory cortices. The spatial profile of EphB3 receptors was co-localized to regions of the brain that had a high level of EphB3 receptor binding ligands. Its expression pattern suggests that EphB3 may play a role in the maintenance of mature neuronal connections or re-arrangement of synaptic connections during late stages of development.

Improved Delivery in Cell Culture of Radiolabeled Antisense DNAs by Duplex Formation

Delivery remains an unresolved problem in applications requiring intravenous administration of DNAs. Recently improved antisense translation interruption in cells was reported for an antisense (AS) oligomer as a duplex compared to singlet AS oligomer presumably because of improved delivery. The unstable phosphodiester backbone of the sense (S) oligomer and its shorter chain length apparently encouraged intracellular dissociation and release of the AS oligomer. We have investigated the mechanism involved to evaluate whether the approach may be useful for antisense radionuclide imaging. Duplexes were formed between an AS phosphorothioate DNA against the mdr1 mRNA and the uniform phoshorothioate or uniform phosphodiester sense (S) DNAs with either four or six mismatches. Accumulations in KB-G2 (Pgp++) cells of radiolabeled AS DNA as duplex accumulated threefold higher compared to singlet. Accumulation was still antisense as shown by reduced accumulations with the radiolabel on the S DNA. However, the DNA backbone had no clear influence on accumulations. Targeting of mRNAs with radiolabeled AS DNAs may be improved in cell culture if duplexed with an S DNA engineered for low hybridization affinity to encourage dissociation in the presence of the target mRNA.

Thermodynamic, Counterion, and Hydration Effects for the Incorporation of Locked Nucleic Acid Nucleotides into DNA Duplexes

A locked nucleic acid (LNA) monomer is a conformationally restricted nucleotide analogue with an extra 2'-O, 4'-C-methylene bridge added to the ribose ring. LNA-modified oligonucleotides are known to exhibit enhanced hybridization affinity toward complementary DNA and RNA. In this work, we have evaluated the hybridization thermodynamics of a series of LNA-substituted DNA octamers, modified to various extents by one to three LNA substitutions, introduced at either adenine (5'-AGCACCAG) or thymine (5'-TGCTCCTG) nucleotides. To understand the energetics, counterion effects, and the hydration contribution of the incorporation of LNA modification, a combination of spectroscopic and calorimetric techniques was used. The CD spectra of the corresponding duplexes showed that the modified duplexes adopt an A-type conformation. UV and DSC melting studies revealed that each type of duplex unfolds in a two-state transition. A complete thermodynamic profile at 5 degrees C indicated that the net effect of modification on thermodynamic parameters might be positional and that the neighboring bases flanking the modification might influence the favorable formation of the modified duplexes. Furthermore, relative to the formation of the unmodified reference duplexes, the formation of modified duplexes is accompanied by a higher uptake of counterions and a lower uptake of water molecules.

Thermodynamics of competitive surface adsorption on DNA microarrays

Gene microarrays provide a powerful functional genomics technology which permits theexpression profiling of tens of thousands of genes in parallel. The basic idea of theirfunctioning is based on the sequence specificity of probe–target interactions combined withfluorescence detection. In reality, this straightforward principle is opposed by thecomplexity of the experimental system due to imperfections of chip fabrication and RNApreparation, due to the non-linearity of the probe response and especially due tocompetitive interactions which are inherently connected with the high throughputcharacter of the method. We theoretically analysed aspects of the hybridization of DNAoligonucleotide probes with a complex multicomponent mixture of RNA fragments,such as the effect of different interactions between nucleotide strands competingwith the formation of specific duplexes, electrostatic and entropic blocking, thefragmentation of the RNA, the incomplete synthesis of the probes and ‘zipping’ effects inthe oligonucleotide duplexes. The effective hybridization affinities of microarrayprobes are considerably smaller than those for bulk hybridization owing to theeffects discussed, but they correlate well with the bulk data on a relative scale. Ingeneral, the hybridization isotherms of microarray probes are shown to deviatefrom a Langmuir-type behaviour. Nevertheless isotherms of the Langmuir or Sipstype are predicted to provide a relatively simple description of the non-linear,probe-specific concentration dependence of the signal intensity of microarrayprobes.

Effective affinities in microarray data

In the past couple of years several studies have shown that hybridization in AffymetrixDNA microarrays can be rather well understood on the basis of simple models ofphysical chemistry. In the majority of the cases a Langmuir isotherm was used to fitexperimental data. Although there is a general consensus about this approach, somediscrepancies between different studies are evident. For instance, some authors havefitted the hybridization affinities from the microarray fluorescent intensities, whileothers used affinities obtained from melting experiments in solution. The formerapproach yields fitted affinities that at first sight are only partially consistentwith solution values. In this paper we show that this discrepancy exists onlysuperficially: a sufficiently complete model provides effective affinities which arefully consistent with those fitted to experimental data. This link provides newinsight on the relevant processes underlying the functioning of DNA microarrays.

An Efficient, Convenient Solid-Phase Synthesis of Amino Acid-Modified Peptide Nucleic Acid Monomers and Oligomers

An efficient and highly versatile method for the synthesis of amino acid-modified peptide nucleic acid (PNA) monomers is described. By using solid-phase Fmoc techniques, such monomers can be assembled readily in a stepwise manner and obtained in high yield with minimal purification. Protected neutral hydrophilic, acidic, and basic amino acids were coupled to 2-chlorotrityl chloride resin. Following Fmoc removal, innovative conditions for the key step, reductive alkylation with N-Fmoc-aminoacetaldehyde, were developed to circumvent problems encountered with previously reported methods. Activation and coupling of pyrimidine and purine nucleobases to the resulting secondary amines afforded amino acid-modified PNA monomers. The mild reaction conditions utilized were compatible with sensitive and labile functional groups, such as tert-butyl ethers and tert-butyl esters. PNA monomers were obtained in 36-42% overall yield and very high purity, after cleavage and purification. Using standard solid-phase Fmoc chemistry, two of these monomers were incorporated with high coupling efficiency into a variety of modified PNA oligomers, including four tetradecamers designed to target bcl-2 mRNA. Such modified oligomers have the potential to enhance water solubility and cell portability, while maintaining hybridization affinity and promoting favorable biodistribution properties.

A General Method for the Synthesis of 2‘-O-Cyanoethylated Oligoribonucleotides Having Promising Hybridization Affinity for DNA and RNA and Enhanced Nuclease Resistance

[reaction: see text] An effective method for the synthesis of 2'-O-cyanoethylated oligoribonucleotides as a new class of 2'-O-modified RNAs was developed. The reaction of appropriately protected ribonucleoside derivatives with acrylonitrile in t-BuOH in the presence of Cs2CO3 gave 2'-O-cyanoethylated ribonucleoside derivatives in excellent yields, which were converted by a successive selective deprotection/protection strategy to 2'-O-cyanoethylated 5'-O-dimethoxytritylribonucleoside 3'-phosphoramidite derivatives in high yields. Fully 2'-O-cyanoethylated oligoribonucleotides, (Uce)12 and (GceAceCceUce)3, were successfully synthesized in the phosphoramidite approach by use of the phosphoramidite building blocks. It was also found that oligoribonucleotides having a 2'-O-cyanoethylated ribonucleoside (Uce, Cce, Ace, or Gce) could be obtained by the selective removal of the TBDMS group from fully protected oligoribonucleotide intermediates without loss of the cyanoethyl group by use of NEt3 x 3HF as a desilylating reagent. The detailed T(m) experiments revealed that oligoribonucleotides containing 2'-O-cyanoethylated ribonucleosides have higher hybridization affinity for both DNA and RNA than the corresponding unmodified and 2'-O-methylated oligoribonucleotides. In addition, introduction of a cyanoethyl group into the 2'-position of RNA resulted in significant increase of nuclease resistance toward snake venom and bovine spleen phosphodiesterases compared with that of the methyl group.

SNPs analysis by use of oligonucleotides containing 2′-O-methyl-2-thiouridines

In this study, we report an improved method for the synthesis of oligonucleotides containing 2-thiouridine (s2U) derivatives. The hybridization affinity and base pair recognition ability of oligonucleotides having 2-thiouracil moieties with both the complementary RNAs and DNAs were also studied in detail. These results indicate that oligonucleotides containing s2U derivatives could be used as powerful tools for chip-based SNPs detection.

Affinity Enhancement Bivalent Morpholinos for Pretargeting: Surface Plasmon Resonance Studies of Molecular Dimensions

Bivalent effectors have been reported to provide superior pretargeting by affinity enhancement. We recently reported that one bivalent MORF (phosphorodiamidate morpholino, a DNA analogue oligomer) exhibited affinity enhancement (ratio of bivalent to monovalent equilibrium constants for binding) to immobilized complementary DNA (cDNA) by surface plasmon resonance (SPR). Because bivalent effectors using oligomers are easily synthesized with molecular spacing between binding sites, an important determinant of binding, adjustable simply by selecting linkers of different dimensions and/or lengthening or shortening the oligomer chain length, they may have advantages over existing bivalent effectors. We synthesized four bivalent MORFs with different dimensions between binding sites and measured binding affinities and affinity enhancement by SPR. An 18 mer (MORF18) was made bivalent by dimerizing both with disuccinimidyl suberate (DSS) and disuccinimidyl glutarate (DSG) linkers. By again using DSS but adding seven nonbinding adenine bases and by eliminating six binding bases, a total of four bivalent effectors, DSS-MORF12, DSG-MORF18, DSS-MORF18, and DSS-MORF25, were prepared with two different hybridization affinities (i.e. MORF12 and MORF18/25) and three different spacings (i.e. 20, 25, and 100 angstroms) between binding sites. The biotinylated cDNA was immobilized on a sensor chip at 500 and 100 RU coating densities providing an average cDNA separation of 25 and 80 angstroms. As expected, bimolecular binding dominated monomolecular binding in all cases, especially at lower MORF effector concentrations and at higher coating densities. All bivalent MORFs showed equilibrium constants superior to their monovalent form and therefore affinity enhancement. DSS-MORF25 showed the highest equilibrium constant for bimolecular binding presumably because of its larger separation between binding sites. Nevertheless, DSS-MORF12 showed the largest affinity enhancement of almost 3 orders of magnitude presumably because the shorter chain lowered the equilibrium constant for monomolecular binding. We have shown that bivalent effectors may be easily synthesized using MORF. The results provide further evidence that the use of bivalent effectors can greatly improve MORF pretargeting and, finally, that bivalent MORFs with reduced equilibrium constants may actually exhibit higher affinity enhancement.