Organocatalytic removal of formaldehyde adducts from RNA and DNA bases

Abstract

Formaldehyde is universally used to fix tissue specimens, where it forms hemiaminal and aminal adducts with biomolecules, hindering the ability to retrieve molecular information. Common methods for removing these adducts involve extended heating, which can cause extensive degradation of nucleic acids, particularly RNA. Here, we show that water-soluble bifunctional catalysts (anthranilates and phosphanilates) speed the reversal of formaldehyde adducts of mononucleotides over standard buffers. Studies with formaldehyde-treated RNA oligonucleotides show that the catalysts enhance adduct removal, restoring unmodified RNA at 37 °C even when extensively modified, while avoiding the high temperatures that promote RNA degradation. Experiments with formalin-fixed, paraffin-embedded cell samples show that the catalysis is compatible with common RNA extraction protocols, with detectable RNA yields increased by 1.5-2.4-fold using a catalyst under optimized conditions and by 7-25-fold compared with a commercial kit. Such catalytic strategies show promise for general use in reversing formaldehyde adducts in clinical specimens.

Figure 1

Formaldehyde adducts and catalysts in this study. (a) Adducts on N6 of adenine (R= ribose or deoxyribose); formaldehyde residues are shown in red. Similar adducts are formed on exocyclic amines of cytosine and guanine. (b) Transimination catalyst structures studied here.

Figure 2

Relative rates of formaldehyde adduct reversal. (a) Relative rates of reversal of the hemiaminal adduct of dAMP in Tris buffer alone (pH 7) or with 10 mM added catalysts shown. Catalysts provide 1.6–4.1 fold rate enhancement over buffer, with the largest change in rate due to bifunctional catalyst 3. (b) Rates of reversal of the aminal crosslink of AMP in phosphate buffer alone (pH 4.5) or with 10 mM added catalysts. Catalysts 1 and 2 show moderate rate enhancements, while catalyst 3 provides a superior 37-fold increase in rate. Data are from three replicates each (error bars are standard deviations).

Figure 3

Relative yields of adduct reversal from nucleotides after 1 h with catalyst 3 and with analogs having functional groups omitted (structures shown). (a) Reversal of the hemiaminal adduct of dAMP (pH 7, 16 mM catalyst, 37 °C); (b) Reversal of the aminal dimer of AMP (pH 4.5, 16 mM catalyst, 37 °C). In both cases the bifunctional catalyst 3 performs better than either of the closely-related monofunctional catalysts. Data are from three replicates each (error bars are standard deviations).

Figure 4

Assessing formaldehyde adducts on an RNA strand by mass spectrometry. (a) Sequence of the self-complementary 16mer RNA, which was designed to promote adducts and crosslinks on unpaired bases. (b) MALDI mass spectrum of formaldehyde-treated RNA, showing extensive adducts after 24 h treatment (up to 14 per strand, see inset) and little or no unmodified RNA (5181 Da) remaining. Unmodified DNA (4294 Da) is spiked in for reference.

Figure 5

Improvement in reversal of RNA formaldehyde adducts after low-temperature incubation in the presence of catalyst 3. RNA pretreated with 10% formaldehyde (see Fig. 4) was used as starting material. (a) MALDI mass spectrum of 16mer RNA oligonucleotide after 18 h treatment with catalyst 3 (8 mM, pH 7, 37 °C), showing major recovered RNA peak. (b) Time course of RNA recovery at pH 7, comparing 60 °C heating in 8 mM Tris buffer () to 37 °C in 8 mM Tris buffer () and 37 °C in 8 mM catalyst 3 (no Tris) (). Higher temperature provides a short-term increase in RNA recovery, but catalyst 3 gives an advantage at the lower temperature where RNA is more stable. Error bars show standard deviation from 5 experiments. (c) Time course of crosslink reversal in dimerized RNA oligonucleotide, following uncrosslinking to monomer RNA by denaturing PAGE. Treatment at pH 4.5, 37 °C with 16 mM catalyst 3 () yields more uncrosslinking than incubation at pH 4.5, 37 °C in 16 mM citrate buffer (), although full reversal of crosslinks is not observed with this substrate. Error bars show standard deviation from 3 or 4 experiments.

Figure 6

Enhancement in recovery of RNAs from formalin-fixed, paraffin-embedded cell specimens using catalyst 3 (20 mM) as compared with different incubation and isolation conditions. Amplifiable RNA yield is plotted for eight amplicons, and quantity is determined with a standard curve. Lane 1 employs incubation conditions from a commercial kit (Qiagen AllPrep^® DNA/RNA FFPE kit), which uses an 80 °C, 0.25 h incubation step without catalyst (“no cat”) and a spin column for isolation, with the addition of catalyst to these conditions shown in lane 2 (“cat. 3”). Optimized incubation conditions (55 °C, 18 h) followed by a spin column RNA isolation are shown in lanes 3–4. A common literature procedure is shown in lane 5 (“PCI”). Addition of the catalyst to the optimized incubation conditions results in a ~2 fold increase in detectable RNA, and more substantial increases relative to the catalyst-free commercial kit protocol (see enhancements in red) or the literature protocol. The means of three independent experiments are shown, error bars indicating the standard deviation of variation in the qRT-PCR yield. SC: spin column isolation. PCI: Masuda protocol of phenol-chloroform-isoamyl alcohol extraction followed by heating in buffer. A.U.: arbitrary units. Significance for pairwise comparisons shown was tested using a 1-tailed paired samples t-test. *: P < 0.05; **: P < 0.01. Fold enhancements are shown below brackets; enhancement relative to the commercial kit is shown in red.