The Role of Key Amino Acids of the Human Fe(II)/2OG-Dependent Dioxygenase ALKBH3 in Structural Dynamics and Repair Activity toward Methylated DNA.

Non-heme dioxygenases of the AlkB family hold a unique position among enzymes that repair alkyl lesions in nucleic acids. These enzymes activate the Fe(II) ion and molecular oxygen through the coupled decarboxylation of the 2-oxoglutarate co-substrate to subsequently oxidize the substrate. ALKBH3 is a human homolog of E. coli AlkB, which displays a specific activity toward N1-methyladenine and N3-methylcytosine bases in single-stranded DNA. Due to the lack of a DNA-bound structure of ALKBH3, the basis of its substrate specificity and structure-function relationships requires further exploration. Here we have combined biochemical and biophysical approaches with site-directed mutational analysis to elucidate the role of key amino acids in maintaining the secondary structure and catalytic activity of ALKBH3. Using stopped-flow fluorescence spectroscopy we have shown that conformational dynamics play a crucial role in the catalytic repair process catalyzed by ALKBH3. A transient kinetic mechanism, which comprises the steps of the specific substrate binding, eversion, and anchoring within the DNA-binding cleft, has been described quantitatively by rate and equilibrium constants. Through CD spectroscopy, we demonstrated that replacing side chains of Tyr143, Leu177, and His191 with alanine results in significant alterations in the secondary structure content of ALKBH3 and decreases the stability of mutant proteins. The bulky side chain of Tyr143 is critical for binding the methylated base and stabilizing its flipped-out conformation, while its hydroxyl group is likely involved in facilitating the product release. The removal of the Leu177 and His191 side chains substantially affects the secondary structure content and conformational flexibility, leading to the complete inactivation of the protein. The mutants lacking enzymatic activity exhibit a marked decrease in antiparallel ß-strands, offset by an increase in the helical component.

Phosphoramidate Azole Oligonucleotides for Single Nucleotide Polymorphism Detection by PCR.

Detection of the Kirsten rat sarcoma gene (KRAS) mutational status is an important factor for the treatment of various malignancies. The most common KRAS-activating mutations are caused by single-nucleotide mutations, which are usually determined by using PCR, using allele-specific DNA primers. Oligonucleotide primers with uncharged or partially charged internucleotide phosphate modification have proved their ability to increase the sensitivity and specificity of various single nucleotide mutation detection. To enhance the specificity of single nucleotide mutation detection, the novel oligonucleotides with four types of uncharged and partially charged internucleotide phosphates modification, phosphoramide benzoazole (PABA) oligonucleotides (PABAO), was used to prove the concept on the KRAS mutation model. The molecular effects of different types of site-specific PABA modification in a primer or a template on a synthesis of full-length elongation product and PCR efficiency were evaluated. The allele-specific PCR (AS-PCR) on plasmid templates showed a significant increase in analysis specificity without changes in Cq values compared with unmodified primer. PABA modification is a universal mismatch-like disturbance, which can be used for single nucleotide polymorphism discrimination for various applications. The molecular insights of the PABA site-specific modification in a primer and a template affect PCR, structural features of four types of PABAO in connection with AS-PCR results, and improvements of AS-PCR specificity support the further design of novel PCR platforms for various biological targets testing.

Properties of phosphoramide benzoazole oligonucleotides (PABAOs). I. Structure and hybridization efficiency of N-benzimidazole derivatives.

In this work, we for the first time conducted a detailed study on the structure, dynamics, and hybridization properties of N-benzimidazole group-bearing phosphoramide benzoazole oligonucleotides (PABAOs) that we developed recently. By circular dichroism we established that the introduction of the modifications does not disrupt the B conformation of the DNA double helix. The formation of complexes is approximated by a two-state model. Complexes of PABAOs with native oligodeoxriboynucleotides form efficiently, and the introduction of such modifications reduces thermal stability of short duplexes (8-10 bp) by ∼5°Ð¡ per modification. Using UV-spectroscopy analysis, a neutral charge of the phosphate residue modified by the N-benzimidazole moiety in the pH range of 3-9.5 was found. The results confirm possible usefulness of PABAOs for both basic research and biomedical applications.

Combined NMR and molecular dynamics conformational filter identifies unambiguously dynamic ensembles of Dengue protease NS2B/NS3pro.

The dengue protease NS2B/NS3pro has been reported to adopt either an 'open' or a 'closed' conformation. We have developed a conformational filter that combines NMR with MD simulations to identify conformational ensembles that dominate in solution. Experimental values derived from relaxation parameters for the backbone and methyl side chains were compared with the corresponding back-calculated relaxation parameters of different conformational ensembles obtained from free MD simulations. Our results demonstrate a high prevalence for the 'closed' conformational ensemble while the 'open' conformation is absent, indicating that the latter conformation is most probably due to crystal contacts. Conversely, conformational ensembles in which the positioning of the co-factor NS2B results in a 'partially' open conformation, previously described in both MD simulations and X-ray studies, were identified by our conformational filter. Altogether, we believe that our approach allows for unambiguous identification of true conformational ensembles, an essential step for reliable drug discovery.

PARP3 Affects Nucleosome Compaction Regulation.

Genome compaction is one of the important subject areas for understanding the mechanisms regulating genes' expression and DNA replication and repair. The basic unit of DNA compaction in the eukaryotic cell is the nucleosome. The main chromatin proteins responsible for DNA compaction have already been identified, but the regulation of chromatin architecture is still extensively studied. Several authors have shown an interaction of ARTD proteins with nucleosomes and proposed that there are changes in the nucleosomes' structure as a result. In the ARTD family, only PARP1, PARP2, and PARP3 participate in the DNA damage response. Damaged DNA stimulates activation of these PARPs, which use NAD+ as a substrate. DNA repair and chromatin compaction need precise regulation with close coordination between them. In this work, we studied the interactions of these three PARPs with nucleosomes by atomic force microscopy, which is a powerful method allowing for direct measurements of geometric characteristics of single molecules. Using this method, we evaluated perturbations in the structure of single nucleosomes after the binding of a PARP. We demonstrated here that PARP3 significantly alters the geometry of nucleosomes, possibly indicating a new function of PARP3 in chromatin compaction regulation.

All Domains of SARS-CoV-2 nsp1 Determine Translational Shutoff and Cytotoxicity of the Protein.

Replication of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strongly affects cellular metabolism and results in rapid development of the cytopathic effect (CPE). The hallmarks of virus-induced modifications are inhibition of translation of cellular mRNAs and redirection of the cellular translational machinery to the synthesis of virus-specific proteins. The multifunctional nonstructural protein 1 (nsp1) of SARS-CoV-2 is a major virulence factor and a key contributor to the development of translational shutoff. In this study, we applied a wide range of virological and structural approaches to further analyze nsp1 functions. The expression of this protein alone was found to be sufficient to cause CPE. However, we selected several nsp1 mutants exhibiting noncytopathic phenotypes. The attenuating mutations were detected in three clusters, located in the C-terminal helices, in one of the loops of the structured domain and in the junction of the disordered and structured fragment of nsp1. NMR-based analysis of the wild type nsp1 and its mutants did not confirm the existence of a stable ß5-strand that was proposed by the X-ray structure. In solution, this protein appears to be present in a dynamic conformation, which is required for its functions in CPE development and viral replication. The NMR data also suggest a dynamic interaction between the N-terminal and C-terminal domains. The identified nsp1 mutations make this protein noncytotoxic and incapable of inducing translational shutoff, but they do not result in deleterious effects on viral cytopathogenicity. IMPORTANCE The nsp1 of SARS-CoV-2 is a multifunctional protein that modifies the intracellular environment for the needs of viral replication. It is responsible for the development of translational shutoff, and its expression alone is sufficient to cause a cytopathic effect (CPE). In this study, we selected a wide range of nsp1 mutants exhibiting noncytopathic phenotypes. The attenuating mutations, clustered in three different fragments of nsp1, were extensively characterized via virological and structural methods. Our data strongly suggest interactions between the nsp1 domains, which are required for the protein's functions in CPE development. Most of the mutations made nsp1 noncytotoxic and incapable of inducing translational shutoff. Most of them did not affect the viability of the viruses, but they did decrease the rates of replication in cells competent in type I IFN induction and signaling. These mutations, and their combinations, in particular, can be used for the development of SARS-CoV-2 variants with attenuated phenotypes.

Synthesis of Oligonucleotides Carrying Inter-nucleotide N-(Benzoazole)-phosphoramide Moieties.

In this work, we present new oligonucleotide derivatives containing inter-nucleotide N-benzimidazole, N-benzoxazole, N-benzothiazole, and 1,3-dimethyl-N-benzimidazole (benzoazoles) phosphoramide groups. These modifications were introduced via the Staudinger reaction with appropriate azides during standard automated solid-phase oligonucleotide synthesis. The principal structural difference between the new azido modifiers and those already known is that they are bulk heterocyclic structures, similar to purine nucleoside bases. Modified oligonucleotides with one and two modifications at different positions and multiple modified heteronucleotide sequences were obtained with high yields. The possibility of multiple modifications in the process of automatic DNA synthesis is fundamental and critical for further application of our oligonucleotide derivatives. Initial studies on the properties of new oligonucleotides were carried out. The stability of the oligodeoxyribonucleotide duplex containing phosphoramide groups of N-benzoazoles with complementary DNA or RNA is slightly lower than that of native complexes.

Allele-Specific PCR for PIK3CA Mutation Detection Using Phosphoryl Guanidine Modified Primers.

Phosphoryl guanidine (PG) is the novel uncharged modification of internucleotide phosphates of oligonucleotides. Incorporating PG modification into PCR primers leads to increased discrimination between wild-type and mutated DNA, providing extraordinary detection limits in an allele-specific real-time polymerase chain reaction (AS-PCR). Herein, we used PG-modification to improve the specificity of AS primers with unfavorable Pyr/Pur primer's 3'-end mismatch in the template/primer complex. Two mutations of the PIK3CA gene (E542K, E545K) were chosen to validate the advantages of the PG modification. Several primers with PG modifications were synthesized for each mutation and assessed using AS-PCR with the plasmid controls and DNA obtained from formalin-fixed paraffin-embedded (FFPE) tissues. The assay allows the detection of 0.5% of mutated DNA on the wild-type DNA plasmid template's background with good specificity. Compared with ddPCR, the primers with PG-modification demonstrated 100% specificity and 100% sensitivity on the DNA from FFPE with mutation presence higher than 0.5%. Our results indicate the high potential of PG-modified primers for point mutation detection. The main principle of the developed methodology can be used to improve the specificity of primers regardless of sequences.

Diversity of Self-Assembled RNA Complexes: From Nanoarchitecture to Nanomachines.

New tool development for various nucleic acid applications is an essential task in RNA nanotechnology. Here, we determined the ability of self-limited complex formation by a pair of oligoribonucleotides carrying two pairwise complementary blocks connected by a linker of different lengths in each chain. The complexes were analyzed using UV melting, gel shift assay analysis, and molecular dynamics (MD) simulations. We have demonstrated the spontaneous formation of various self-limited and concatemer complexes. The linear concatemer complex is formed by a pair of oligomers without a linker in at least one of them. Longer linkers resulted in the formation of circular complexes. The self-limited complexes formation was confirmed using the toehold strand displacement. The MD simulations indicate the reliability of the complexes' structure and demonstrate their dynamics, which increase with the rise of complex size. The linearization of 2D circular complexes into 1D structures and a reverse cyclization process were demonstrated using a toehold-mediated approach. The approach proposed here for the construction and directed modification of the molecularity and shape of complexes will be a valuable tool in RNA nanotechnology, especially for the rational design of therapeutic nucleic acids with high target specificity and the programmable response of the immune system of organisms.

Structure- and Content-Dependent Efficiency of Cas9-Assisted DNA Cleavage in Genome-Editing Systems.

Genome-editing systems, being some of the key tools of molecular biologists, represent a reasonable hope for progress in the field of personalized medicine. A major problem with such systems is their nonideal accuracy and insufficient selectivity. The selectivity of CRISPR-Cas9 systems can be improved in several ways. One efficient way is the proper selection of the consensus sequence of the DNA to be cleaved. In the present work, we attempted to evaluate the effect of formed non-Watson-Crick pairs in a DNA duplex on the efficiency of DNA cleavage in terms of the influence of the structure of the formed partially complementary pairs. We also studied the effect of the location of such pairs in DNA relative to the PAM (protospacer-adjacent motif) on the cleavage efficiency. We believe that the stabilization of the Cas9-sgRNA complex with a DNA substrate containing noncomplementary pairs is due to loop reorganization in the RuvC domain of the enzyme. In addition, PAM-proximal mismatches in the DNA substrate lower enzyme efficiency because the "seed" region is involved in binding and cleavage, whereas PAM-distal mismatches have no significant impact on target DNA cleavage. Our data suggest that in the case of short duplexes with mismatches, the stages of recognition and binding of dsDNA substrates by the enzyme determine the reaction rate and time rather than the thermodynamic parameters affected by the "unwinding" of DNA. The results will provide a theoretical basis for predicting the efficiency and accuracy of CRISPR-Cas9 systems at cleaving target DNA.

Thermodynamic Swings: How Ideal Complex of Cas9-RNA/DNA Forms.

Most processes of the recognition and formation of specific complexes in living systems begin with collisions in solutions or quasi-solutions. Then, the thermodynamic regulation of complex formation and fine tuning of complexes come into play. Precise regulation is very important in all cellular processes, including genome editing using the CRISPR-Cas9 tool. The Cas9 endonuclease is an essential component of the CRISPR-Cas-based genome editing systems. The attainment of high-specificity and -efficiency Cas9 during targeted DNA cleavage is the main problem that limits the practical application of the CRISPR-Cas9 system. In this study, we analyzed the thermodynamics of interaction of a complex's components of Cas9-RNA/DNA through experimental and computer simulation methods. We found that there is a small energetic preference during Cas9-RNA/DNA formation from the Cas9-RNA and DNA/DNA duplex. The small difference in binding energy is relevant for biological interactions and could be part of the sequence-specific recognition of double-stranded DNA by the CRISPR-Cas9 system.

Synthesis of the new nucleoside 5'-alpha-iminophosphates using Staudinger reaction.

Efficient protocols were developed for the synthesis of a new compounds - nucleoside 5'-α-iminophosphates using the Staudinger reaction. These substances are alpha-phosphate mimetic nucleotide in which an oxygen atom is replaced by a corresponding imino (=N-R)-group. Various 5'-iminomonophosphates of nucleosides were obtained. A chemical method for the synthesis of triphosphate derivatives based on the iminomonophosphates has been designed. Thymidine 5'-(1,3-dimethylimidazolidin-2-ylidene)-triphosphate (ppp(DMI)T) was synthesized, its hydrolytic stability and substrate properties in relation to some DNA polymerases was firstly studied. It was shown that ppp(DMI)T can serve as substrate for enzyme catalyzed template-independent DNA synthesis by human terminal deoxynucleotidyltransferase TdT.

Pairing nanoarchitectonics of oligodeoxyribonucleotides with complex diversity: concatemers and self-limited complexes.

The development of approaches to the design of two- and three-dimensional self-assembled DNA-based nanostructures with a controlled shape and size is an essential task for applied nanotechnology, therapy, biosensing, and bioimaging. We conducted a comprehensive study on the formation of various complexes from a pair of oligonucleotides with two transposed complementary blocks that can be linked through a nucleotide or non-nucleotide linker. A methodology is proposed to prove the formation of a self-limited complex and to determine its molecularity. It is based on the "opening" of a self-limited complex with an oligonucleotide that effectively binds to a duplex-forming block. The complexes assembled from a pair of oligonucleotides with different block length and different linker sizes and types were investigated by theoretical analysis, several experimental methods (a gel shift assay, atomic force microscopy, and ultraviolet melting analysis), and molecular dynamics simulations. The results showed a variety of complexes formed by only a pair of oligonucleotides. Self-limited associates, concatemer complexes, or mixtures thereof can arise if we change the length of a duplex and loop-forming blocks in oligonucleotides or via introduction of overhangs and chemical modifications. We postulated basic principles of rational design of native self-limited DNA complexes of desired structure, shape, and molecularity. Our foundation makes self-limited complexes useful tools for nanotechnology, biological studies, and therapeutics.

Structure and hybridization properties of phosphoryl guanidine oligonucleotides under crowding conditions.

Phosphoryl guanidine oligonucleotides (PGOs) are promising uncharged analogs of nucleic acids and are used in a variety of applications. The importance of hydration is frequently ignored during the design of modified nucleic acid probes. Such hydrophobic modifications (phosphoryl guanidine) are expected to have a significant impact on the structure and thermal stability of the affected oligo with complementary nucleic acids. Here we aimed to investigate (by the osmotic stress method) hydration changes upon the formation of a duplex of a PGO with complementary DNA. According to our results, the presence of phosphoryl guanidines in one or both strands of a duplex only minimally affects hydration alterations under crowding conditions. The secondary structure of native and modified duplexes did not change significantly in the presence of ethanol, ethylene glycol, polyethylene glycol 200, or polyethylene glycol 1000. After the addition of a cosolvent, the thermodynamic stability of the PGO complexes changed in the same manner as that seen in a corresponding DNA duplex. The findings reported here and our previous studies form the basis for efficient use of PGOs in basic research and a variety of applications.

Effects of Phosphoryl Guanidine Modification of Phosphate Residues on the Structure and Hybridization of Oligodeoxyribonucleotides.

Phosphoryl guanidine oligonucleotides (PGOs) are promising tools for biological research and development of biosensors and therapeutics. We performed structural and hybridization analyses of octa-, deca-, and dodecamers with all phosphate residues modified by 1,3-dimethylimidazolidine-2-imine moieties. Similarity of the B-form double helix between native and modified duplexes was noted. In PGO duplexes, we detected a decrease in the proportion of C2'-endo and an increased proportion of C1'-exo sugar conformations of the modified chain. Applicability of the two-state model to denaturation transition of all studied duplexes was proved for the first time. Sequence-dependent effects of this modification on hybridization properties were observed. The thermal stability of PGO complexes is almost native at 100 mM NaCl and slightly increases with decreasing ionic strength. An increase in water activity and dramatic changes in interaction with cations and in solvation of PGOs and their duplexes were noted, resulting in slight elevation of the melting temperature after an ionic-strength decrease from 1 M NaCl down to deionized water. Decreased binding of sodium ions and decreased water solvation were documented for PGOs and their duplexes. In contrast to DNA, the PGO duplex formation leads to a release of several cations. The water shell is significantly more disordered near PGOs and their complexes. Nevertheless, changes in solvation during the formation of native and PGO complexes are similar and indicate that it is possible to develop models for predictive calculations of the thermodynamic properties of phosphoryl guanidine oligomers. Our results may help devise an approach for the rational design of PGOs as novel improved molecular probes and tools for many modern methods involving oligonucleotides.

Terminal Mono- and Bis-Conjugates of Oligonucleotides with Closo-Dodecaborate: Synthesis and Physico-Chemical Properties.

Oligonucleotide conjugates with boron clusters have found applications in different fields of molecular biology, biotechnology, and biomedicine as potential agents for boron neutron capture therapy, siRNA components, and antisense agents. Particularly, the closo-dodecaborate anion represents a high-boron-containing residue with remarkable chemical stability and low toxicity, and is suitable for the engineering of different constructs for biomedicine and molecular biology. In the present work, we synthesized novel oligonucleotide conjugates of closo-dodecaborate attached to the 5'-, 3'-, or both terminal positions of DNA, RNA, 2'-O-Me RNA, and 2'-F-Py RNA oligomers. For their synthesis, we employed click reaction with the azido derivative of closo-dodecaborate. The key physicochemical characteristics of the conjugates have been investigated using high-performance liquid chromatography, gel electrophoresis, UV thermal melting, and circular dichroism spectroscopy. Incorporation of closo-dodecaborate residues at the 3'-end of all oligomers stabilized their complementary complexes, whereas analogous 5'-modification decreased duplex stability. Two boron clusters attached to the opposite ends of the oligomer only slightly influence the stability of complementary complexes of RNA oligonucleotide and its 2'-O-methyl and 2'-fluoro analogs. On the contrary, the same modification of DNA oligonucleotides significantly destabilized the DNA/DNA duplex but gave a strong stabilization of the duplex with an RNA target. According to circular dichroism spectroscopy results, two terminal closo-dodecaborate residues cause a prominent structural rearrangement of complementary complexes with a substantial shift from the B-form to the A-form of the double helix. The revealed changes of key characteristics of oligonucleotides caused by incorporation of terminal boron clusters, such as the increase of hydrophobicity, change of duplex stability, and prominent structural changes for DNA conjugates, should be taken into account for the development of antisense oligonucleotides, siRNAs, or aptamers bearing boron clusters. These features may also be used for engineering of developing NA constructs with pre-defined properties.

Strict conformational demands of RNA cleavage in bulge-loops created by peptidyl-oligonucleotide conjugates.

Potent knockdown of pathogenic RNA in vivo is an urgent health need unmet by both small-molecule and biologic drugs. 'Smart' supramolecular assembly of catalysts offers precise recognition and potent destruction of targeted RNA, hitherto not found in nature. Peptidyl-oligonucleotide ribonucleases are here chemically engineered to create and attack bulge-loop regions upon hybridization to target RNA. Catalytic peptide was incorporated either via a centrally modified nucleotide (Type 1) or through an abasic sugar residue (Type 2) within the RNA-recognition motif to reveal striking differences in biological performance and strict structural demands of ribonuclease activity. None of the Type 1 conjugates were catalytically active, whereas all Type 2 conjugates cleaved RNA target in a sequence-specific manner, with up to 90% cleavage from 5-nt bulge-loops (BC5-α and BC5L-ß anomers) through multiple cuts, including in folds nearby. Molecular dynamics simulations provided structural explanation of accessibility of the RNA cleavage sites to the peptide with adoption of an 'in-line' attack conformation for catalysis. Hybridization assays and enzymatic probing with RNases illuminated how RNA binding specificity and dissociation after cleavage can be balanced to permit turnover of the catalytic reaction. This is an essential requirement for inactivation of multiple copies of disease-associated RNA and therapeutic efficacy.

Allele-Specific PCR for KRAS Mutation Detection Using Phosphoryl Guanidine Modified Primers.

Establishing the Kirsten rat sarcoma (KRAS) mutational status is essential in terms of managing patients with various types of cancer. Allele-specific real-time polymerase chain reaction (AS-PCR) is a widely used method for somatic mutations detection. To improve the limited sensitivity and specificity, several blocking methods have been introduced in AS-PCR to block the amplification of wild-type templates. Herein, we used a novel modified oligonucleotide with internucleotide phosphates reshaped 1,3-dimethyl-2-imino-imidazolidine moieties (phosphoryl guanidine (PG) groups) as primers and blockers in the AS-PCR method. Four common KRAS mutations were chosen as a model to demonstrate the advantages of the PG primers and blockers utilizing a customized PCR protocol. The methods were evaluated on plasmid model systems providing a KRAS mutation detection limit of 20 copies of mutant DNA in a proportion as low as 0.1% of the total DNA, with excellent specificity. PG-modification can serve as the universal additional mismatch-like disturbance to increase the discrimination between wild-type and mutated DNA. Moreover, PG can serve to increase primer specificity by a synergetic effect with additional mismatch and would greatly facilitate medical research.

G-quadruplex 2'-F-modified RNA aptamers targeting hemoglobin: Structure studies and colorimetric assays.

Biosensors that rely on aptamers as analyte-recognizing elements (also known as aptasensors) are gaining in popularity during recent years for analytical and biomedical applications. Among them, colorimetric ELISA-like systems seem very promising for biomarker detection in medical diagnostics. For their development, one should thoroughly consider the characteristics of the aptamers, with a particular focus on the secondary structure. In this study, we performed an in-depth structural study of previously selected hemoglobin-binding 2'-F-RNA aptamers using CD spectroscopy, enzymatic probing, and specific fluorophore binding. Only a combination of different assays allowed us to prove G-quadruplex formation for anti-hemoglobin 2'-F-RNA aptamers. We also demonstrated a possible application of these 2'-F-RNA aptamers for microplate colorimetric detection of human hemoglobin in both direct and sandwich formats.

Postsynthetic On-Column 2' Functionalization of RNA by Convenient Versatile Method.

We report a universal straightforward strategy for the chemical synthesis of modified oligoribonucleotides containing functional groups of different structures at the 2' position of ribose. The on-column synthetic concept is based on the incorporation of two types of commercial nucleotide phosphoramidites containing orthogonal 2'-O-protecting groups, namely 2'-O-thiomorpholine-carbothioate (TC, as "permanent") and 2'-O-tert-butyl(dimethyl)silyl (tBDMS, as "temporary"), to RNA during solid-phase synthesis. Subsequently, the support-bound RNA undergoes selective deprotection and follows postsynthetic 2' functionalization of the naked hydroxyl group. This convenient method to tailor RNA, utilizing the advantages of solid phase approaches, gives an opportunity to introduce site-specifically a wide range of linkers and functional groups. By this strategy, a series of RNAs containing diverse 2' functionalities were synthesized and studied with respect to their physicochemical properties.