Origins of the enhanced affinity of RNA-protein interactions triggered by RNA phosphorodithioate backbone modification


The well-characterized interaction between the MS2 coat protein and its cognate RNA hairpin was used to evaluate changes in affinity as a result of phosphorodithioate (PS2) replacing phosphate by biolayer interferometry (BLI). A structure-based analysis of the data provides insights into the origins of the enhanced affinity of RNA-protein interactions triggered by the PS2 moiety.

Selection of PD1/PD-L1 X-Aptamers


Specific, chemically modified aptamers (X-Aptamers) were identified against two immune checkpoint proteins, recombinant Programmed Death 1 (PD-1) and Programmed Death Ligand 1 (PD-L1). Selections were performed using a bead-based X-Aptamer (XA) library containing several different amino acid functional groups attached to dU at the 5-position. The binding affinities and specificities of the selected XA-PD1 and XA-PDL1 were validated by hPD-1 and hPD-L1 expression cells, as well as by binding to human pancreatic ductal adenocarcinoma tissue. The selected PD1 and PDL1 XAs can mimic antibody functions in in vitro assays.


X-Aptamer Selection and Validation


Aptamers and second generation analogs, such as X-Aptamers (XAs), SOMAmers, locked nucleic acids (LNAs), and others are increasingly being used for molecular pathway targeting, biomarker discovery, or disease diagnosis by interacting with protein targets on the surface of cells or in solution. Such targeting is being used for imaging, diagnostic evaluation, interference of protein function, or delivery of therapeutic agents. Selection of aptamers using the original SELEX method is cumbersome and time-consuming, often requiring 10–15 rounds of selection, and provides aptamers with a limited number of functional groups, namely four bases of DNA or RNA, although newer SELEX methods have increased this diversity. In contrast, X-Aptamers provide an unlimited number of functional groups and thus are superior targeting agents. Here, we discuss the X-Aptamer selection process.

RNA Nanostructures pp 151-174. Part of the Methods in Molecular Biology book series (MIMB, volume 1632). 21 July 2017.

Nano-SPRi Aptasensor for the Detection of Progesterone in Buffer.


Progesterone is a steroid hormone that plays a central role in the female reproductive processes such as ovulation and pregnancy with possible effects on other organs as well. The measurement of progesterone levels in bodily fluids can assist in early pregnancy diagnosis and can provide insight for other reproductive functions. In this work, the detection of progesterone was examined by integrating novel aptamer development with a nanoEnhanced surface plasmon resonance imaging sensor. First, we developed X-aptamers and selected them for binding to progesterone. Then, we took advantage of the multi-array feature of SPRi to develop an optimized biosensor capable of simultaneously screening the 9 X-aptamers developed to determine the binding capabilities of each aptamer. The sensor surface design conditions were further optimized for the sandwich assay, which employed nanoEnhancers (NIR-streptavidin coated quantum dots) for ultrasensitive detection of progesterone molecules. The assay designed was examined over a concentration range of 1.575 ng/mL to 126 μg/mL resulting in a limit of detection (LOD) of 1.575 ng/mL (5 nM) in phosphate buffer.

Sci Rep 2016, 6, 26714.

Electrochemical aptamer scaffold biosensors for detection of botulism and ricin toxins.


Protein toxins present considerable health risks, but detection often requires laborious analysis. Here, we developed electrochemical aptamer biosensors for ricin and botulinum neurotoxins, which display robust and specific signal at nanomolar concentrations and function in dilute serum. These biosensors may aid future efforts for the rapid diagnosis of toxins.

Chem Commun (Camb) 2015, 51, 15137-40.

X-aptamers: a bead-based selection method for random incorporation of druglike moieties onto next-generation aptamers for enhanced binding.


By combining pseudorandom bead-based aptamer libraries with conjugation chemistry, we have created next-generation aptamers, X-aptamers (XAs). Several X-ligands can be added in a directed or random fashion to the aptamers to further enhance their binding affinities for the target proteins. Here we describe the addition of a drug (N-acetyl-2,3-dehydro-2-deoxyneuraminic acid), demonstrated to bind to CD44-HABD, to a complete monothioate backbone-substituted aptamer to increase its binding affinity for the target protein by up to 23-fold, while increasing the drug’s level of binding 1-million fold.

Biochemistry 2012, 51, 8321-3.

Construction and selection of bead-bound combinatorial oligonucleoside phosphorothioate and phosphorodithioate aptamer libraries designed for rapid PCR-based sequencing.


Chemically synthesized combinatorial libraries of unmodified or modified nucleic acids have not previously been used in methods to rapidly select oligonucleotides binding to target biomolecules such as proteins. Phosphorothioate oligonucleotides (S‐ODNs) or phosphorodithioate oligonucleotides (S2‐ODNs) with sulfurs replacing one or both of the non‐bridging phosphate oxygens bind to proteins more tightly than unmodified oligonucleotides and have the potential to be used as diagnostic reagents and therapeutics. We have applied a split synthesis methodology to create one‐bead one‐S‐ODN and one‐bead one‐S2‐ODN libraries. Binding and selection of specific beads to the transcription factor NF‐κB p50/p50 protein were demonstrated. Sequencing both the nucleic acid bases and the positions of any 3′‐O‐thioate/dithioate linkages was carried out by using a novel PCR‐based identification tag of the selected beads. This approach allows us to rapidly and conveniently identify S‐ODNs or S2‐ODNs that bind to proteins.

Nucleic Acids Res 2002, 30, e132.

Evoking picomolar binding in RNA by a single phosphorodithioate linkage.


RNA aptamers are synthetic oligonucleotide-based affinity molecules that utilize unique three-dimensional structures for their affinity and specificity to a target such as a protein. They hold the promise of numerous advantages over biologically produced antibodies; however, the binding affinity and specificity of RNA aptamers are often insufficient for successful implementation in diagnostic assays or as therapeutic agents. Strong binding affinity is important to improve the downstream applications. We report here the use of the phosphorodithioate (PS2) substitution on a single nucleotide of RNA aptamers to dramatically improve target binding affinity by ∼1000-fold (from nanomolar to picomolar). An X-ray co-crystal structure of the α-thrombin:PS2-aptamer complex reveals a localized induced-fit rearrangement of the PS2-containing nucleotide which leads to enhanced target interaction. High-level quantum mechanical calculations for model systems that mimic the PS2 moiety and phenylalanine demonstrate that an edge-on interaction between sulfur and the aromatic ring is quite favorable, and also confirm that the sulfur analogs are much more polarizable than the corresponding phosphates. This favorable interaction involving the sulfur atom is likely even more significant in the full aptamer-protein complexes than in the model systems.

Crystal structure, stability and Ago2 affinity of phosphorodithioate-modified RNAs


Small interfering RNAs (siRNAs) with phosphorodithioate modifications (PS2-RNA) possess favourable properties for use as RNAi therapeutics. Beneficial here is the combining of PS2 and 20 -O-methyl modifications (MePS2). SiRNAs with MePS2 moieties in the sense strand show promising efficacies in vitro and in vivo. Crystal structures of PS2- and MePS2- modified RNAs reveal subtle changes in geometry and hydration compared with natural RNA. A model of an MePS2- RNA–PAZ domain complex points to a hydrophobic effect as the source of the higher affinity of MePS2-RNA for Ago2.

RSC Adv., 2014, 4, 64901

2′-OMe-phosphorodithioate-modified siRNAs show increased loading into the RISC complex and enhanced anti-tumour activity.


Improving small interfering RNA (siRNA) efficacy in target cell populations remains a challenge to its clinical implementation. Here, we report a chemical modification, consisting of phosphorodithioate (PS2) and 2′-O-Methyl (2′-OMe) MePS2 on one nucleotide that significantly enhances potency and resistance to degradation for various siRNAs. We find enhanced potency stems from an unforeseen increase in siRNA loading to the RNA-induced silencing complex, likely due to the unique interaction mediated by 2′-OMe and PS2. We demonstrate the therapeutic utility of MePS2 siRNAs in chemoresistant ovarian cancer mouse models via targeting GRAM domain containing 1B (GRAMD1B), a protein involved in chemoresistance. GRAMD1B silencing is achieved in tumours following MePS2-modified siRNA treatment, leading to a synergistic anti-tumour effect in combination with paclitaxel. Given the previously limited success in enhancing siRNA potency with chemically modified siRNAs, our findings represent an important advance in siRNA design with the potential for application in numerous cancer types.




4-6 Weeks from Target Receipt to Material Shipped


Higher Affinity Binding


Extensive Modifications Available

No PCR Bias

Raptamer Discovery Group’s mission is to help you find great binding molecules for your research or commercial application. Faster, stronger, better: the Raptamer™ way!

Let’s Discover a Raptamer™ Together!