ABSTRACT
A practical fluorescence-labeling method for sequencing small RNAs by the traditional `direct read out' on polyacrylamide gel
electrophoresis was established. The 3
'
terminus of RNA was oxidized into dialdehyde by sodium periodate and then
labeled with fluorescein-5-thiosemicarbazide through the condensation reaction between
carbazide and aldehyde. The fluorescence-labeled RNA was partially degraded enzymatically and fractionated by
polyacrylamide gel electrophoresis. The fluorescent bands were visualised by
ultraviolet photography. A partial sequence of yeast 5S rRNA was determined.
The result indicates that this method can be used in sequencing small RNAs
rapidly, conveniently and safely.
In the early 1980s, Liu
et al.
proposed sequencing RNA by fluorescence-labeling, and sequenced an oligoribonucleotide by step-wise degradation (
9
). A practical fluorescence-labeling method for sequencing small RNA by the traditional `direct read out' on
polyacrylamide gel electrophoresis has now been
established and used to determine the sequence of the 3' terminal 69 nucleotides (nt) of yeast 5S rRNA. The result is identical
to the known sequence. This indicates that the fluorescence-labeling method can be used in sequencing small RNAs rapidly, conveniently
and safely.
In this method, sodium periodate was used to oxidize the 3' terminus of RNA into dialdehyde. The excess of the oxidant was removed
by adding sodium sulfite, and then the fluorescent dye, fluorescein-5-thiosemicarbazide, was added to label the 3' terminus of RNA through the condensation reaction between
carbazide and aldehyde. The detailed procedure is as follows: 5 [mu]g yeast 5S rRNA was dissolved in 10 [mu]l redistilled water, then 10 [mu]l of buffer (0.25 M sodium acetate, pH 5.6) and sodium periodate was
added. The molar ratio of RNA and sodium periodate was 1/10 in a final volume
of 40 [mu]l. The oxidization of 3' terminus of RNA was carried out at 25oC in the dark for 90 min. Then a 2-fold excess of sodium sulphite over sodium periodate was
added to the system to remove the excess oxidant. The reaction mixture was
incubated at 25oC for 15 min. Finally, fluorescein-5-thiosemicarbazide was added; the molar ratio of the fluorescent
dye to the RNA was 30:1. After labeling for 3 h at 37oC in the dark, the RNA was precipitated by adding 1/10 vol of 8 M LiCl and
2.5 vol ethanol, standing at -20oC for 3 h, and then centrifuged (13 000
g
at 4oC) for 20 min. The precipitate was washed with 75% ethanol several times to
remove the free fluorescent dye and the labeled RNA was used for sequence
determination. In these operations, no obvious degradation of RNA was observed.
The sequence of the fluorescence-labeling RNA was analyzed by enzymatic degradation as the same mainly as
described for sequencing 5' or 3' terminal
32
P-labeled RNA (
10
). Partial digestion of the terminally labeled RNA with base-specific ribonucleases T
1
, U
2
,
Bacillus cerus
or
Phy
I is performed at elevated temperature (50-55oC) and in the presence of 7 M urea in the cases of ribonucleases T
1
and U
2
to avoid interference of the secondary/tertiary structure of RNA on enzymatic
hydrolysis. Then the labeled RNA fragments, generated by the partial digestion
of 3' terminal labeled RNA, were separated according to their chain length by
15% polyacrylamide gel electrophoresis (containing 15% DMF for short RNA
fragments).
An ultraviolet detecting device with short wavelength of ultraviolet light was
constructed to detect the fluorescent bands in the gel. The total power of the
ultraviolet light was 24 W and an ultraviolet glass plate of the detecting
device was 20 * 15 cm. The ultraviolet glass plate was cleaned with redistilled water
and kept wet. As soon as the electrophoresis finished, the glass plate on one
side of the gel was taken away and the gel was peeled off carefully from the
glass plate and put directly on the ultraviolet glass plate. The gel was kept
smooth on the surface of the ultraviolet glass plate with no intervening air
bubbles. After exciting with short wavelength ultraviolet light and exposing a
film for 8 min by a camera with color filter and black-and-white film insensitive to ultraviolet light, clear and sharp bands
were obtained on the film (Fig.
1
). The sensitivity of a single band of the fluorescent RNA fragment is <0.01 pmol.
Among the methods used in determining the primary structure of RNA, the
classical procedure established by Holley
et al.
involves the identification of nucleotides by their ultraviolet absorption
spectra, which is more time-consuming and usually requires large amounts of purified RNA (
1
). The prelabeling techniques developed by Sanger and his colleagues, suitable
for the study of
32
P-labeled RNA, have great advantages both in the separation of
oligoribonucleotide fragments and in the sensitivity of detection (
2
). Later, several postlabeling techniques for sequencing RNA by high resolution
polyacrylamide gel electrophoresis were developed. These techniques include
enzymatic digestion (
3
), chemical degradation (
4
,
5
) and the wandering-spot method (
6
). These methods are highly sensitive but need the use of radioisotopes and are
also labor-intensive, time-consuming and fairly expensive. Since fluorescence-labeling for sequencing nucleic acid avoids the use of
radioisotopes, many researchers have developed fluorescent DNA sequencing (
7
,
8
) which is widely used. However, no similar fluorescent method for sequencing
RNA has been reported yet.
REFERENCES
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