Directed Evolution of Aptamer Discovery Technologies

Although antibodies are a powerful tool for molecular biology and clinical diagnostics, there are many emerging applications for which nucleic acid-based aptamers can be advantageous. However, generating high-quality aptamers with sufficient affinity and specificity for biomedical applications is a challenging feat for most research laboratories. In this Account, we describe four techniques developed in our lab to accelerate the discovery of high quality aptamer reagents that can achieve robust binding even for challenging molecular targets. The first method is particle display, in which we convert solution-phase aptamers into aptamer particles that can be screened via fluorescence-activated cell sorting (FACS) to quantitatively isolate individual aptamer particles based on their affinity. This enables the efficient isolation of high-affinity aptamers in fewer selection rounds than conventional methods, thereby minimizing selection biases and reducing the emergence of artifacts in the final aptamer pool. We subsequently developed the multi-parametric particle display (MPPD) method, which employs two-color FACS to isolate aptamer particles based on both affinity and specificity, yielding aptamers that exhibit excellent target binding even in complex matrices like serum. The third method is a click chemistry-based particle display (click-PD) that enables the generation and high-throughput screening of non-nattural aptamers with a wide range of base modifications. We have shown that these base-modified aptamers can achieve robust affinity and specificity for targets that have proven challenging or inaccessible with natural nucleotide-based aptamer libraries. Lastly, we describe the non-natural aptamer array (N2A2) platform, in which a modified benchtop sequencing instrument is used to characterize base-modified aptamers in a massively parallel fashion, enabling the efficient identification of molecules with excellent affinity and specificity for their targets. This system first generates aptamer clusters on the flow-cell surface that incorporate alkyne-modified nucleobases, and then performs a click reaction to couple those nucleobases to an azide-modified chemical moiety. This yields a sequence-defined array of tens of millions of base-modified sequences, which can then be characterized in a high-throughput fashion. Collectively, we believe that these advancements are helping to make aptamer technology more accessible, efficient, and robust, thereby enabling the use of these affinity reagents for a wider range of molecular recognition and detection-based applications.

Non-Canonical Helical Structure of Nucleic Acids Containing Base-Modified Nucleotides

Chemically modified nucleobases are thought to be important for therapeutic purposes as well as diagnosing genetic diseases and have been widely involved in research fields such as molecular biology and biochemical studies. Many artificially modified nucleobases, such as methyl, halogen, and aryl modifications of purines at the C8 position and pyrimidines at the C5 position, are widely studied for their biological functions. DNA containing these modified nucleobases can form non-canonical helical structures such as Z-DNA, G-quadruplex, i-motif, and triplex. This review summarizes the synthesis of chemically modified nucleotides: (i) methylation, bromination, and arylation of purine at the C8 position and (ii) methylation, bromination, and arylation of pyrimidine at the C5 position. Additionally, we introduce the non-canonical structures of nucleic acids containing these modifications.

Pan-cancer analysis of m5C regulator genes reveals consistent epigenetic landscape changes in multiple cancers

Background 5-Methylcytosine (m5C) is a reversible modification to both DNA and various cellular RNAs. However, its roles in developing human cancers are poorly understood, including the effects of mutant m5C regulators and the outcomes of modified nucleobases in RNAs. Methods Based on The Cancer Genome Atlas (TCGA) database, we uncovered that mutations and copy number variations (CNVs) of m5C regulatory genes were significantly correlated across many cancer types. We then assessed the correlation between the expression of individual m5C regulators and the activity of related hallmark pathways of cancers. Results After validating m5C regulators’ expression based on their contributions to cancer development and progression, we observed their upregulation within tumor-specific processes. Notably, our research connected aberrant alterations to m5C regulatory genes with poor clinical outcomes among various tumors that may drive cancer pathogenesis and/or survival. Conclusion Our results offered strong evidence and clinical implications for the involvement of m5C regulators.

Pan-cancer analysis of m5C regulator genes reveals consistent epigenetic landscape changes in multiple cancers

Backgroud5-methylcytosine (m5C) is a reversible modification to both DNA and various cellular RNAs. However, its roles in developing human cancers are poorly understood, including the effects of mutant m5C regulators and the outcomes of modified nucleobases in RNAs. Methods Based on The Cancer Genome Atlas (TCGA) database, we uncovered that mutations and copy number variations (CNVs) of m5C regulatory genes were significantly correlated across 33 cancer types. We then assessed the correlation between the expression of individual m5C regulators and the activity of related hallmark pathways of cancers. After validating m5C regulators' expression based on their contributions to cancer development and progression, we observed their up-regulation within tumor-specific processes. Results Notably, our research connected aberrant alterations to m5C regulatory genes with poor clinical outcomes among various tumors that may drive cancer pathogenesis and/or survival. Conclusions Our results offered strong evidence and clinical implications for the involvement of m5C regulators.

The effect of selenium, zinc and copper on the excretion of urinary modified nucleobases in rats treated with prostate cancer cells

Given the strong associations between diet and cancer risk, there is considerable scientific interest in determining whether dietary factors associated with prostate cancer cell implantation may influence epigenetic alternations. The aim of the research was to assess impact of selected trace elements (selenium, zinc and copper) on the kinetics of changes (10-13-14-21 week of life cycle of rats) in the level of 7-methylguanine, 3-methyladenine, 1-methylguanine and 8-oxo-guanine in the urine of rats with implanted prostate cancer cells (LNCaP). Modified nucleobases were determined by validated high performance liquid chromatography coupled to mass spectrometry (LC-MS/MS) method using multiple reaction monitoring (MRM) mode. In the presented model the implantation of rats with cancer cells did not affect the level of the examined biomarkers in the rats’ urine. The level of methyl derivatives was statistically significantly reduced with the age of the examined rats. The implantation of rats with cancer cells results in the appearance of tumors in 71% of the rats obtaining the standard diet and respectively in 25% of those supplemented with selenium. Supplementation with selenium affects both the effectiveness of tumor induction and the concentration of 7-MeG, 3-MeA, 1-MeG and 8-oxoG in urine of the examined rats. These findings show that modified nucleosides can play an important role in cancer prevention.

Fluorescent Biaryl Uracils with C5-Dihydro- and Quinazolinone Heterocyclic Appendages in PNA

There has been much effort to exploit fluorescence techniques in the detection of nucleic acids. Canonical nucleic acids are essentially nonfluorescent; however, the modification of the nucleobase has proved to be a fruitful way to engender fluorescence. Much of the chemistry used to prepare modified nucleobases relies on expensive transition metal catalysts. In this work, we describe the synthesis of biaryl quinazolinone-uracil nucleobase analogs prepared by the condensation of anthranilamide derivatives and 5-formyluracil using inexpensive copper salts. A selection of modified nucleobases were prepared, and the effect of methoxy- or nitro- group substitution on the photophysical properties was examined. Both the dihydroquinazolinone and quinazolinone modified uracils have much larger molar absorptivity (~4–8×) than natural uracil and produce modest blue fluorescence. The quinazolinone-modified uracils display higher quantum yields than the corresponding dihydroquinazolinones and also show temperature and viscosity dependent emission consistent with molecular rotor behavior. Peptide nucleic acid (PNA) monomers possessing quinazolinone modified uracils were prepared and incorporated into oligomers. In the sequence context examined, the nitro-substituted, methoxy-substituted and unmodified quinazolinone inserts resulted in a stabilization (∆Tm = +4.0/insert; +2.0/insert; +1.0/insert, respectively) relative to control PNA sequence upon hybridization to complementary DNA. All three derivatives responded to hybridization by the “turn-on” of fluorescence intensity by ca. 3-to-4 fold and may find use as probes for complementary DNA sequences.

Thermochemical and Structural Analysis of Tautomers of Sulfur and Selenium Modified RNA Nucleobases

Nucleobases (adenine, cytosine, guanine, and uracil), the four molecules that forms RNA, have been found to be useful in probing in the human body when modified because they can emit light. Non-modified nucleobases do not exhibit emissive properties and cannot be used as probes. Some of the modifications include the substitution of nitrogen atoms with sulfur and selenium, and the resulting modified nucleobases give place to the so-called tz- and ts- RNA alphabets, respectively. Therefore, the aim of this project was to provide insights about the viability, from a computational perspective, of using the modified nucleobases as probes, evaluating the differences in thermochemical, structural and emissive properties of the modified nucleobases with respect to the non-modified ones. Nucleobases can coexist with other modified nucleobases or tautomers, molecules that differ due to the change in position of hydrogen atoms in a molecule’s structure and as a result have different physical and chemical properties. The thermochemical properties evaluation mainly consisted in the computation of the relative Gibbs Free Energy (G), which is related to the fraction F, an index of the relative population among tautomers. This was done using Gaussian 09 software by performing geometry analysis and frequency computations on each one of the tautomers. By comparing the equilibrium fractions, it was determined that in both cases, tz- and ts- guanine and cytosine exist principally in the form of one of their tautomers (Cytosine 2 and Guanine 2) as in the case of the non-modified cases. After confirming which tz- and ts- tautomers were the ones with the largest probable population, infrared (IR) and ultraviolet-visible (UV-vis) spectra were obtained. The IR spectra of selenium and sulfur tautomers of guanine and cytosine indicated that the tautomers had peaks at similar frequencies with respect to each other, however, the intensities varied, implying slight structural changes between the tautomers. On the other hand, the UV-vis spectra showed a change in peak positions between the tautomers with sulfur and selenium, suggesting that the change between sulfur and selenium has an effect on the spectra by shifting the peaks from the original molecules’ λmax values. Their relative population fractions show that only the canonical forms of the modified nucleobases exist in a larger extent than the rest of their tautomer forms. In addition, the features in their UV-vis and IR spectra allow these tautomers to be differentiated from each other.