Base pairing and structural insights into the 5-formylcytosine in RNA duplex.

5-Formylcytidine (f(5)C), a previously discovered natural nucleotide in the mitochondrial tRNA of many species including human, has been recently detected as the oxidative product of 5-methylcytidine (m(5)C) through 5-hydroxymethylcytidine (hm(5)C) in total RNA of mammalian cells. The discovery indicated that these cytosine derivatives in RNA might also play important epigenetic roles similar as in DNA, which has been intensively investigated in the past few years. In this paper, we studied the base pairing specificity of f(5)C in different RNA duplex contexts. We found that the 5-formyl group could increase duplex thermal stability and enhance base pairing specificity. We present three high-resolution crystal structures of an octamer RNA duplex [5'-GUA(f(5)C)GUAC-3']2 that have been solved under three crystallization conditions with different buffers and pH values. Our results showed that the 5-formyl group is located in the same plane as the cytosine base and forms an intra-residue hydrogen bond with the amino group in the N4 position. In addition, this modification increases the base stacking between the f(5)C and the neighboring bases while not causing significant global and local structure perturbations. This work provides insights into the effects of 5-formylcytosine on RNA duplex.

Conjugation and Evaluation of Triazole-Linked Single Guide RNA for CRISPR-Cas9 Gene Editing.

The CRISPR-Cas9 gene editing system requires Cas9 endonuclease and guide RNAs (either the natural dual RNA consisting of crRNA and tracrRNA or a chimeric single guide RNA) that direct site-specific double-stranded DNA cleavage. This communication describes a click ligation approach that uses alkyne-azide cycloaddition to generate a triazole-linked single guide RNA (sgRNA). The conjugated sgRNA shows efficient and comparable genome editing activity to natural dual RNA and unmodified sgRNA constructs.

Boron-containing aptamers to ATP.

Boron neutron capture therapy (BNCT), an experimental treatment for certain cancers, destroys only cells near the boron; however, there is a need to develop highly specific delivery agents. As nucleic acid aptamers recognize specific molecular targets, we investigated the influence of boronated nucleotide analogs on RNA function and on the systematic evolution of ligands by exponential enrichment (SELEX) process. Substitution of guanosine 5'-(alpha-P-borano) triphosphate (bG) for GTP or uridine 5'-(alpha-P-borano) triphosphate (bU) for UTP in several known aptamers diminished or eliminated target recognition by those RNAs. Specifically, ATP-binding aptamers containing the zeta-fold, which appears in several selections for adenosine aptamers, became inactive upon bG substitution but were only moderately affected by bU substitution. Selections were carried out using the bG or bU analogs with C8-linked ATP agarose as the binding target. The selections with bU and normal NTP yielded some zeta-fold aptamers, while the bG selection yielded none of this type. Non-zeta aptamers from bU and bG populations tolerated the borano substitution and many required it. The borano nucleotide requirement is specific; bU could not be used in bG-dependent aptamers nor vice versa. The borano group plays an essential role, as yet undefined, in target recognition or RNA structure. We conclude that the bG and bU nucleotides are fully compatible with SELEX, and that these analogs could be used to make boronated aptamers as therapeutics for BNCT.

Versatility of chemically synthesized guide RNAs for CRISPR-Cas9 genome editing.

The CRISPR-Cas9 system has become the most popular and efficient method for genome engineering in mammalian cells. The Streptococcus pyogenes Cas9 nuclease can function with two types of guide RNAs: the native dual crRNA and tracrRNA (crRNA:tracrRNA) or a chimeric single guide RNA (sgRNA). Although sgRNAs expressed from a DNA vector are predominant in the literature, guide RNAs can be rapidly generated by chemical synthesis and provide equivalent functionality in gene editing experiments. This review highlights the attributes and advantages of chemically synthesized guide RNAs including the incorporation of chemical modifications to enhance gene editing efficiencies in certain applications. The use of synthetic guide RNAs is also uniquely suited to genome-scale high throughput arrayed screening, particularly when using complex phenotypic assays for functional genomics studies. Finally, the use of synthetic guide RNAs along with DNA-free sources of Cas9 (mRNA or protein) allows for transient CRISPR-Cas9 presence in the cell, thereby resulting in a decreased probability of off-target events.

Reading, writing, and modulating genetic information with boranophosphate mimics of nucleotides, DNA, and RNA.

The P-boranophosphates are efficient and near perfect mimics of natural nucleic acids in permitting reading and writing of genetic information with high yield and accuracy. Substitution of a borane (-BH3) group for oxygen in the phosphate ester bond creates an isoelectronic and isosteric mimic of natural nucleotide phosphate esters found in mononucleotides, i.e., AMP and ATP, and in RNA and DNA polynucleotides. Compared to natural nucleic acids, the boranophosphate RNA and DNA analogs demonstrate increased lipophilicity and resistance to endo- and exonucleases, yet they retain negative charge and similar spatial geometry. Borane groups can readily be introduced into the NTP and dNTP nucleic acid monomer precursors to produce alpha-P-borano nucleoside triphosphate analogs (e.g., NTPalphaB and dNTPalphaB). The NTPalphaB and dNTPalphaB are, in fact, good to excellent substrates for RNA and DNA polymerases, respectively, and allow ready enzymatic synthesis of RNA and DNA with P-boranophosphate linkages. Further, boranophosphate polymer products are good templates for replication, transcription, and gene expression; boronated RNA products are also suitable for reverse transcription to cDNA. Fully substituted boranophosphate DNA can activate the RNase H cleavage of RNA in RNA:DNA hybrids. Moreover, certain dideoxy-NTPalphaB analogs appear to be better substrates for viral reverse transcriptases than the regular ddNTPs, and may offer promising prodrug alternatives in antiviral therapy. These properties make boranophosphates promising candidates for diagnostics; aptamer selection; gene therapy; and antiviral, antisense, and RNAi therapeutics. The boranophosphates constitute a versatile family of phosphate mimics for processing genetic information and modulating gene function.

Synthesis and Separation of Diastereomers of Ribonucleoside 5'-(alpha-P-Borano)triphosphates.

Nucleoside boranophosphates, in which one of the phosphate oxygens is replaced by a borane group, are isoionic and isoelectronic analogues of naturally occurring nucleotides. Boranophosphates also are biochemically important congeners of phosphorothioates and methylphosphonates. We have developed a convenient one-pot method to synthesize the set of ribonucleoside (A, U, G, and C) 5'-(alpha-P-borano)triphosphates. Phosphitylation of the 2',3'-protected ribonucleoside with 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one gives the 5'-phosphite intermediate 2 which undergoes in situ substitution in the presence of pyrophosphate to give the cyclic intermediate, P(2),P(3)-dioxo-P(1)-ribonucleosidylcyclotriphosphate 3. Immediate oxidation of compound 3 with amine.borane complex results in ribonucleoside 5'-(alpha-P-borano)cyclotriphosphate 4. Subsequent reaction of compound 4 with water followed by ammonium hydroxide yields the crude product as a diastereomeric mixture of ribonucleoside 5'-(alpha-P-borano)triphosphate 6. Pure compound 6 is isolated in 30-45% overall yield using ion-exchange chromatography. The separation of two diastereomers of ribonucleoside 5'-(alpha-P-borano)triphosphate 6 is achieved by reverse phase HPLC.