Biomedical subjects

Ponzy Lu

Publications and source records attributed to Ponzy Lu.

7 recordsLinked to original sources

PubMed2005

Stemless self-quenching reporter molecules identify target sequences in mRNA.

The design of oligonucleotides for gene silencing requires a rational method for identifying hybridization-accessible sequences within the target RNA. To this end, we have developed stem-loop self-quenching reporter molecules (SQRMs) as probes for such sequence. SQRMs have a 5' fluorophore, a quenching moiety on the 3' end, an intervening sequence that forms an approximately 5-basepaired stem, and a loop sequence of approximately 20-30 bases. We have previously described a mapping strategy employing SQRMs to locate stem-loop structures in the target mRNA molecule. We now show that the original design constraint of a basepaired stem is not needed, either in vitro or in vivo. We propose that stemless probes possess sufficient signal-to-noise for use in vivo and that this ratio is an indication of hybridization of the probe to its target. Data showing that these SQRMs can specifically target and reduce c-Myb protein synthesis and can be used for real-time in vivo assays are presented.

Animals↗

PubMed2005

Cooperativity of paired oligonucleotide probes for microarray hybridization assays.

Synthetic DNA probes attached to microarrays usually range in length from 25 to 70 nucleotides. There is a compromise between short probes with lower sensitivity, which can be accurately synthesized in higher yields, and long probes with greater sensitivity but lower synthesis yields. Described here are microarrays printed with spots containing a mixture of two short probes, each designed to hybridize at noncontiguous sites in the same targeted sequence. We have shown that, for a printed microarray, mixed probe spots containing a pair of 30mers show significantly greater hybridization than spots containing a single 30mer and can approach the amount of hybridization to spots containing a 60mer or a 70mer. These spots with mixed oligonucleotide probes display cooperative hybridization signals greater than those that can be achieved by either probe alone. Both the higher synthesis yields of short probes and the greater sensitivity of long oligonucleotides can be utilized. This strategy provides new design options for microarray hybridization assays to detect RNA abundance, RNA splice variants, or sequence polymorphisms.

Bacillus subtilis↗

PubMed2005

Identification of antisense nucleic acid hybridization sites in mRNA molecules with self-quenching fluorescent reporter molecules.

We describe a physical mRNA mapping strategy employing fluorescent self-quenching reporter molecules (SQRMs) that facilitates the identification of mRNA sequence accessible for hybridization with antisense nucleic acids in vitro and in vivo, real time. SQRMs are 20-30 base oligodeoxynucleotides with 5-6 bp complementary ends to which a 5' fluorophore and 3' quenching group are attached. Alone, the SQRM complementary ends form a stem that holds the fluorophore and quencher in contact. When the SQRM forms base pairs with its target, the structure separates the fluorophore from the quencher. This event can be reported by fluorescence emission when the fluorophore is excited. The stem-loop of the SQRM suggests that SQRM be made to target natural stem-loop structures formed during mRNA synthesis. The general utility of this method is demonstrated by SQRM identification of targetable sequence within c-myb and bcl-6 mRNA. Corresponding antisense oligonucleotides reduce these gene products in cells.

Animals↗

PubMed2004

Oxetane modified, conformationally constrained, antisense oligodeoxyribonucleotides function efficiently as gene silencing molecules.

Incorporation of nucleosides with novel base-constraining oxetane (OXE) modifications [oxetane, 1-(1',3'-O-anhydro-beta-d-psicofuranosyl nucleosides)] into antisense (AS) oligodeoxyribonucleotides (ODNs) should greatly improve the gene silencing efficiency of these molecules. This is because OXE modified bases provide nuclease protection to the natural backbone ODNs, can impart T(m) values similar to those predicted for RNA-RNA hybrids, and not only permit but also accelerate RNase H mediated catalytic activity. We tested this assumption in living cells by directly comparing the ability of OXE and phosphorothioate (PS) ODNs to target c-myb gene expression. The ODNs were targeted to two different sites within the c-myb mRNA. One site was chosen arbitrarily. The other was a 'rational' choice based on predicted hybridization accessibility after physical mapping with self-quenching reporter molecules (SQRM). The Myb mRNA and protein levels were equally diminished by OXE and PS ODNs, but the latter were delivered to cells with approximately six times greater efficiency, suggesting that OXE modified ODNs were more potent on a molar basis. The rationally targeted molecules demonstrated greater silencing efficiency than those directed to an arbitrarily chosen mRNA sequence. We conclude that rationally targeted, OXE modified ODNs, can function efficiently as gene silencing agents, and hypothesize that they will prove useful for therapeutic purposes.

Cell Division↗

PubMed2002

Role of hydration in the binding of lac repressor to DNA.

The osmotic stress technique was used to measure changes in macromolecular hydration that accompany binding of wild-type Escherichia coli lactose (lac) repressor to its regulatory site (operator O1) in the lac promoter and its transfer from site O1 to nonspecific DNA. Binding at O1 is accompanied by the net release of 260 +/- 32 water molecules. If all are released from macromolecular surfaces, this result is consistent with a net reduction of solvent-accessible surface area of 2370 +/- 550 A. This area is only slightly smaller than the macromolecular interface calculated for a crystalline repressor dimer-O1 complex but is significantly smaller than that for the corresponding complex with the symmetrical optimized O(sym) operator. The transfer of repressor from site O1 to nonspecific DNA is accompanied by the net uptake of 93 +/- 10 water molecules. Together these results imply that formation of a nonspecific complex is accompanied by the net release of 165 +/- 43 water molecules. The enhanced stabilities of repressor-DNA complexes with increasing osmolality may contribute to the ability of Escherichia coli cells to tolerate dehydration and/or high external salt concentrations.

Acetamides↗

PubMed2002

Determination of DNA structure in solution: enzymatic deuteration of the ribose 2' carbon.

An enzymatic solution to the problem of obtaining 13C/15N-labeled nucleotides that are deuterated uniquely at the H2' ' position within the ribose ring is presented. Selective deuteration occurs with an overall yield of >80%. The deuteron at the H2' ' position allows measurement of the scalar and residual dipolar couplings for the bond vectors attached to the C2' carbon of each ribose sugar. These data allow the accurate determination of sugar conformation. Interesting DNA double helices of 2-3 turns are now within the reach of solution NMR spectroscopy. As an example, these labeled nucleotides are incorporated uniquely at positions 6-14 in a 20-bp DNA sequence containing the adenovirus major late promoter.

Carbon Isotopes↗

PubMed2002

Residual dipolar couplings in nucleic acid structure determination.

Solution NMR spectroscopy of nucleic acids has been limited by the short-range nature of the nuclear Overhauser effect and scalar coupling restraints normally used in structure determination. The addition of residual dipolar couplings, obtained from slightly oriented mixtures, provides bond vector angles relative to a universal alignment tensor. The accurate determination of helix curvature, domain orientation and the stoichiometry of homomultimeric nucleic acid complexes is now possible.

Magnetic Resonance Spectroscopy↗