By modifying nucleotides from either or both the stem and loop of precursor-miRNA, greater specificity is achieved as to the mRNAs targeted. The improved efficiency in target regulation can be either mediated by direct base-pairings between targets and precursor miRNAs or indirectly by energy constraints on the availability of such base-pairings. It is found that pri- and pre-miRNA are active in the absence of functional mature miRNA, so pri- or pre-miRNA or truncated portion thereof, but not as mature miRNA, can be used as RNAi agents by themselves. Furthermore, besides the seed sequence of the stem of the pre-miRNA and 3′-extensions thereof to provide greater complementarity to the target mRNA, nucleotides in the loop can affect the activity and specificity of the precursor-miRNA and the processing and binding to target mRNA. By using mutated sequences in the natural pri- or pre-miRNA or modified mimetics, one can screen for target mRNA, target a unique mRNA or a group of mRNAs for regulation, and modulate cell properties with greater specificity and investigate cellular activity as to phenotype and response to external stimuli in the presence or absence of at least partial target protein expression.